^- 


THE  LIBRARY 

OF 

THE  UNIVERSITY 

OF  CALIFORNIA 

LOS  ANGELES 


GIFT  OF 


Dr.  Emil  Bogen 


VACCINE  AND  SERUM  THERAPY 

Including  also  a  Study  of  Infections,   Theories 
OF  Immunity,  Opsonins  and  the 
Opsonic  Index 


EDWIN  HENRY  SCHORER,  B.  S.,  M.  D., 

Assistant  Professor  of  Parasitology  and  Hygiene,  University  of  Missouri; 

FORMERLY    ASSISTANT    ROCKEFELLER    INSTITUTE    FOR    MeDICAL 

Research,  New  York  City. 


ILLUSTRATED. 


St.  Louis 

C.  V.  MOSBY  CO. 

1909. 


Copyrighted  by  C.  V.  Mosby  Co.,  1909. 


iiiomedicaj  , 

Libtaxj 

I^Of  CONTENTS. 


I.  INFECTIONS.  Page 

Course  of  infection 7 

II.  IMMUNITY. 

Natural  and  acquired.      Active  and  passive 12 

Theories  of  immunity:  Klebs  and  Pasteur,  Chauveau,  Metchnikoff,  Salmon 
and  Smith,  Fodor,  Behring  and  Knorr,  Ehrlich's  side  chain  theory;  opsonin 
theory,  Wright  and  Douglas;  Neufeld  and  Rimpau 14 

III.  OPSONIC  INDEX. 

Leishman's  method  of  estimating  phagocytic  power 23 

Wright  and  Douglas'  opsonic  index 23 

Serum;  leucocytes;  bacterial  emulsion;  strength  of  bacterial  emulsion;  mix- 
ture of  bacteria,  leucocytes  and  serum;  smears,  preparation,  fixing  and 
staining;  examination  of  smears;  calculation  of  the  opsonic  index 24 

IV.  CRITICISMS    AND    MODIFICATIONS    OF    WRIGHT'S    OPSONIC    INDEX 
DETERMINATIONS. 

Mechanical  technique;  serum;  bacterial  emulsions;  leucocytes;  calculation 
and  determination  of  the  opsonic  index,  Simon,  Strong;  leucocj^tes  ex- 
amined     37 

V.  OPSONIC  INDEX  IN  HEALTH  AND  DISEASE. 

Importance  of  opsonins  in  immunity  and  accuracy  of  the  method  of  deter- 
mining the  opsonic  index;  changes  in  opsonic  index  in  disease;  changes  in 
opsonic  index  due  to  vaccines 49 

VI.  NATURE  OF  OPSONINS. 

Specificity;  effect  of  heat;  structure  of  opsonins;  relation  to  leucocytosis;  rela- 
tion to  stimulins;  opsonins  and  aggressins;  influence  of  chemicals  and 
reaction;  non-bacterial  opsonins 58 

VII.  VACCINE  THERAPY. 

Bacterial  Vaccines:    dosage;  organisms  used  in  vaccination;    preparation  of 

vaccines;  control  of  injections,  opsonic  index  and  clinical 64 

Staphylococcus,  streptococcus,  gonococcus,  pneumococcus,  tuberculosis,  colon 
Bacillus,  Bacillus  typhosus.  Bacillus  dysenteriae,  cholera,  and  less  com- 
mon infections 73 

Specific  vaccines  for  diseases  of  unknown  etiologj'':    hydrophobia,  small-pox.  ...    81 

Future  of  opsonins  and  vaccines 85 

VIII.  SERUM  THERAPY. 

Historical:  Untoward  effects  of  serum  injections,  serum  disease  and  In'per- 
susceptibility  to  serum;  indications  for  immune  serum  injections;  con- 
centration and  purification  of  serum;  dried  immune  serum;  oral  adminis- 
tration of  serum 87 

Anti-toxic  serum:     Diphtheria  antitoxin;  tetanus  anti-toxin 99 

Anti-bacterial  serum:  Anti-streptococcic  syrum;  anti-meningococcic  serum; 
anti-gonococcic  serum;  anti-pneumococcic  serum;  anti-typhoid  serum; 
anti-dysenteric  serum;  anti-staphylococcic  serum 109 


fi^fif).Q5 


PREFACE 


Treatment  of  individual  diseases  with  medicines  or  b}^  methods  having 
a  selective  curative  action  has  until  recently  been  limited.  With  the  establish- 
ment of  the  germ  theory  of  certain  diseases  and  the  development  of  informa- 
tion concerning  immunity,  new  methods  of  specific  treatment  were  made  pos- 
sible and  are  now  practiced  under  the  name  of  serum  and  vaccine  therapy. 
As  might  be  expected,  the  medical  profession  has  been  much  interested  in  these 
methods  of  treatment,  and  applied  them  whenever  possible.  The  develop- 
ment of  vaccine  and  serum  therapy  has  been  slow,  the  methods  have  had  to 
be  revised  and  in  some  cases  the  results  obtained  have  been  found  to  be  other- 
wise than  was  at  first  expected.  Because  of  this,  much  confusion  has  arisen. 
The  practitioner  has  not  been  able  to  keep  pace  with  the  developments  and 
literature  on  these  subjects,  and  finally  has  been  forced  to  depend  on  the  state- 
ments and  recommendations  coming  from  serum  and  vaccine  laboratories, 
enthusiasts  and  ever  exploiters. 

In  this  work  an  attempt  has  been  made  to  state  concisely  and  accurately 
the  present  knowledge  concerning  vaccines  and  immune  sera.  An  effort  has 
been  made  to  establish  theoretical  and  experimental  evidence  as  well  as  clin- 
ical application  of.  the  specific  treatment  of  bacterial  diseases.  To  accomplish 
this,  some  space  is  given  to  infections  in  general,  the  theories  of  immunity, 
with  especial  emphasis  on  the  opsonic  theory  of  immunity,  as  well  as  the  par- 
ticular methods  of  vaccine  and  serum  therapy. 

Considerable  space  has  been  given  to  opsonins,  the  opsonic  index  and  the 
importance  of  opsonins  in  health  and  disease.  This  has  been  done  because 
since  1904  no  subject  lias  appeared  more  prominently  or  frequently  in  medical 
literature  than  that  concerning  opsonins,  opsonic  immunity,  and  bacterial 
vaccines.  In  the  first  presentations  of  discoveries  on  this  form  of  immunity 
and  specific  treatment  of  bacterial  diseases,  great  possibilities  were  promised. 
Methods,  which  would,  according  to  Wright,  give  uniform  success  in  treat- 
ment of  the  large  class  of  bacterial  infections  and  diseases,  naturally  received 
immediate  and  general  attention  by  the  medical  Avorld  and  were  at  once  quite 
generally  applied.  This  has  been  followed  b}-  much  indiscriminate  and  un- 
scientific use  of  tliese  methods  of  specific  treatment  so  that  in  the  minds    of 


mail}-,  opsonins  and  vaccine  therapy,  have  gone  into  disrepute  as  did  tuber- 
culin neaily  twenty  years  ago.  We  now  know  tuberculin  has  many  appli- 
cations of  importance  in  the  diagnosis  and  treatment  of  tuberculosis,  though 
this  knowledge  lias  been  gained  some  years  later  than  it  would  have  been  had 
it  not  been  for  improper  exploitation.  In  the  hope  of  avoiding  a  repetition 
of  such  an  effect  as  far  as  opsonin  and  bacterial  vaccines  are  concerned,  this 
subject  is  given  considerable  attention.  The  opsonic  index  technique  which 
is  given  here  is  the  one  taught  the  writer  by  Dr.  W.  G.  Ross,  who  was  for  two 
years  a  pupil  of  Sir  A.  E.  Wright. 

An  attempt  has  been  made  to  bring  the  su])jects  taken  up  as  nearly  up 
to  date  as  is  possible.  It  is  hoped  that  this  work  may  furnish  to  the  medical 
student  and  practitioner  information  which  may  lead  him  to  a  better  under- 
standing of  the  nature  of  infections  and  the  subjects  of  immunit}^,  and  active 
and  passive  immunization. 


Columbia,  Missouri,  April,  1909. 


CHAPTER  I. 


INFECTIONS 


By  the  term  infection  we  understand  the  entrance  of  micro- 
parasitic  living  agents  into  the  body  tissue  or  substance,  and 
the  occurence  of  definite  symptoms  of  disease  as  the  restdt  of 
the  multiplication  and  action  of  the  invading  organisms.  Infec- 
tions do  not  always  occur  when  pathogenic  microorganisms  are 
present  in  the  body  tissues,  certain  conditions  being  necessary 
for  the  establishment  of  a  bacterial  disease.  These  conditions 
may  be  broadly  divided  into  two  classes;  those  dependent  upon 
the  biological  properties  of  the  infecting  organism,  and  those 
dependent  upon  the  conditions  in  the  host  and  tissues  invaded. 

Of  the  biological  characters  of  importance  in  the  infecting 
organism  in  the  production  of  a  bacterial  disease,  the  most  im- 
portant are,  that  the  microorganisms  must  be  able  to  multiply 
rapidly  and  greatly  in  the  tissues  of  the  body,  and  that  they  must 
be  able  to  produce  poisons  or  substances  harmful,  either  to  some 
or  to  all,  of  the  tissues  of  the  bod}'.  The  number  of  organisms 
necessarv'  to  cause  disease  varies  with  different  species.  Many 
more  staphylococci  are  necessary  to  cause  the  formation  of  a 
furuncle  than  of  anthrax  bacilli  to  cause  anthrax.  The  property 
to  produce  substances  poisonous  to  the  body  tissues  generally 
determines  virulence.  That  virulence  is  of  great  importance  is 
evident  from  the  fact  that  innumerable  so-called  saprophytic 
bacteria  which  are  present  in  the  different  parts  of  the  body,  grow 
and  multiply  there  but  because  in  their  growth  sufficient  poison 
is  not  produced,  cannot  cause  an  infection.  The  virulence  of  an 
organism,  according  to  numerous  investigators,  depends  upon 
the  presence  of  certain  substances  in  the  parasite  which  reduce 
the  resistance  of  the  body  and  its  tissues.  These  substances  are 
various!}'  designated  as  lysins  and  aggressins. 


8  VACCINE    AND    SERUM   THERAPY. 

Besides  conditions  in  the  causal  organism,  certain  conditions 
must  exist  in  the  host  or  body  so  that  infection  can  occur.  Cer- 
tain animals  are  insusceptible  to  the  action  of  certain  species  of 
microorganism,  thus  for  example,  the  horse  and  other  domestic 
animals  are  naturally  immune  to  venereal  diseases.  Again  cer- 
tain organisms  can  only  produce  disease  when  they  are  present 
in  certain  parts  of  the  body  and  have  entered  the  body  by  certain 
portals  of  entry.  The  spirillum  cholerae  can  only  produce  its 
typical  form  of  disease  when  it  has  gained  entrance  through  the 
small  intestine.  Invasion  of  the  dermis  or  epidermis  by  this  or- 
ganism produces  no  disease.  The  bacilli  producing  tuberculosis  as 
well  as  certain  other  organisms,  can  cause  infection  when  the}- 
have  entered  through  the  skin  or  through  the  mucous  membrane. 
Certain  organisms  as  Bact.  diphtheriae,  B.  tuberculosis,  and  B. 
typhosus,  show  predilections  for  certain  tissues  but  will  also  pro- 
duce diseased  conditions  in  other  parts  of  the  body. 

In  addition  to  the  non-susceptibility  of  the  host,  the  body 
possesses  certain  natural  barriers  to  disease  which  must  be  over- 
come before  infections  can  be  produced.  The  unbroken  skin 
usually  offers  a  barrier  to  infection.  Age,  sex,  race,  occupation, 
etc. ,  at  times  account  for  certam  resistance  to  invasions  of  organ- 
isms. The  resistance  furthermore  varies  for  the  different  organ- 
isms, as  is  evidenced  by  the  fact  that  for  B.  tetanus  a  wound 
is  necessaiy  to  produce  lock-jaw,  while  for  the  glanders  bacillus 
the  slightest  abrasion  of  the  muccus  membrane  will  furnish  a 
focus  for  an  infection.  The  body  fluids,  lymph  glands,  phagocytes, 
all  offer  resistance  to  infection.  These  barriers  are  overcome  in 
various  ways  and  under  different  conditions,  so  that  while  indi- 
viduals may  be  immune  at  one  time  they  may,  at  another  time, 
be  susceptible  to  the  same  infectious  agent. 


COURSE  OF  INFECTIONS. 


From  the  foregoing  it  is  evident  that  not  only  must  the  in- 
fectious organism  come  into  contact  with  the  body  tissues  to 
produce  disease,  but  the  organism  must  be  able  to  grow,  multi- 
ply, produce  its  poison,  and  overcome  the  resistance  of  a  body 
susceptible  to  its  action.  The  symptoms  and  signs  of  infections 
do  not  appear  until  a  certain  time  after  the  invasion  b\'  the  micro- 


INFECTIONS.  9 

organism.  This  period  is  known  as  the  "incubation  period,"  and 
varies  according  to  the  biological  characters  of  the  infecting  or- 
ganism, but  is  also  influenced  by  the  number  and  virulence  of 
the  organisms  and  the  individual  susceptibility  of  the  host. 

The  course  of  a  disease  is  determined  by  conditions  produced 
by  the  specific  organism  and  partly  by  the  distribution  of  these 
organisms.  But  even  here  it  is  to  be  noted  that  the  course  of 
the  disease  varies.  Many  of  these  variations  we  are  by  no  means 
able  to  explain. 

The  symptoms  and  signs  produced  by  an  organism  will  vaiy 
as  the  organism  acts  locally  or  generally.  In  local  infections 
the  most  marked  disturbance  occurs  at  the  portal  of  entiy  of  the 
microorganism,  while  in  general  infections  the  reaction  manifests 
itself  in  all,  or  a  large  part,  of  the  body.  The  infecting  microor- 
ganism may  produce  disease  either  in  a  mechanical  wa}^  because 
of  large  numbers,  or,  as  is  the  case  of  most  all  infections,  because 
of  toxins  which  may  act  locally  or  be  distributed  over  a  large  part 
or  the  whole,  of  the  body.  Some  organisms,  as  the  bacilli  of  diph- 
theria and  of  tetanus,  produce  extracellular  toxins,  while  other 
organisms,  as  those  causing  typhoid  fever  and  cholera,  have 
intracellular  toxins  which  are  liberated  supposed^  when  the 
organisms  disintegrate. 

After  organisms  have  entered  the  body  the  distribution  varies 
with  the  species.  Thus  we  see  that  staphylococci  form  furuncles, 
carbuncles  and  pustules  in  localized  areas,  typhoid  bacilli  though 
causing  lesions  in  the  intestine  are  usually  present  in  the  blood 
stream,  while  the  tetanus  bacilli  form  a  toxin  which  is  distributed 
through  the  entire  body.  At  times  microorganisms  do  not  remain 
localized  at  the  portal  of  entry  but  pass  on  to  the  lymphatic 
glands  and  other  parts  of  the  body,  here  producing  pathological 
conditions.  In  some  infections  the  microorganisms  gradually 
involve  more  and  more  of  the  tissues  of  the  body.  Microorgan- 
isms may  first  lodge  in  one  part  of  the  body  and  produce  disease 
in  that  part  and  from  these  primary  lesions,  other  parts  of  the 
body  may  become  infected.  When  the  blood  stream  is  not  onh^ 
the  canier  of  microorganisms  but  becomes  the  place  for  gro\^i:h 
and  reproduction  of  the  same,  a  septicaemia  arises.  Wliile  almost 
any  of  these  conditions  may  be  produced  by  any  of  the  pathogenic 
bacteria,  still  certain  organisms  are  more  Hkely  to  produce  one 


10  VACCINE    AND    SKRUM    THERAPY. 

or  another  of  these  conditions.  Resultant  of  this  tendency  of 
microorganisms  to  produce  particular  kinds  of  conditions,  the 
prognosis  for  infections  varies  with  the  species  of  microorganism 
])roducing  the  lesions  or  condition. 

The  same  species  of  microorganism  will  not  always  be  distri- 
buted in  an  identical  manner;  the  location  of  the  lesion  and  the 
condition  produced  var\dng  with  the  properties  of  the  particular 
strain  in  question  and  the  particular  part  of  the  body  infected. 
The  appearance  of  general  symptoms  will  vary  greatly  with  the 
rapidity  of  the  absolution  of  the  toxin.  Experimentally,  intra- 
venous and  intraperitoneal  injections  of  microorganisms  are  fol- 
lowed by  symptoms  earlier  and  more  consistently  than  are  sub- 
cutaneous injections. 

The  number  of  bacteria  invading  the  tissues  is  of  some  im- 
portance. If  few  bacteria  are  present  they  mav  die  without 
producing  infections,  while  if  larger  numbers  are  present  some 
of  them  will  be  likely  to  grow^  and  produce  disease. 

At  the  point  of  infection,  or  portal  of  entry,  the  effects  pro- 
duced will  var\^  with  the  infecting  organism,  some  producing 
specific  forms  of  inflammation  along  with  those  common  to  many 
bacteria.  Even  with  the  same  species  of  microorganism  the 
reaction  will  vary  with  the  prevalence  and  virulence  of  the  invad- 
ing organism  and  with  the  anatomical  structure  of  the  part  of 
the  body  involved.  The  local  reaction,  however,  is  not  only  pro- 
duced when  the  bacteria  enter  the  tissues  of  the  body  but  also 
when  the  toxins  produced  by  the  microorganism  are  present  in 
the  tissues.  The  most  commonly  seen  reaction  at  the  portal  of 
entry  is  that  which  results  in  pus  formation,  in  which,  due  to 
chemotaxis,  leucocytes  accumulate.  While  a  local  reaction  is 
usually  produced  at  the  portal  of  entr}^  of  bacteria,  the  reaction 
at  times  may  be  slight  or  absent  entirely.  A  local  reaction  when 
produced  is  generally  regarded  as  of  importance  in  the  protection 
of  the  body  against  the  invasion  of  bacteria. 

Besides  the  local  reaction,  general  reactions  usually  follow 
in  all  severe  infections.  General  reactions  occur  as  a  result  of 
the  action  of  toxins  produced  by  the  microorganisms  when  they 
are  absorbed  by  body  tissues.  The  general  symptoms  produced 
vary  according  to  the  location  of  the  primary  lesion,  the  extent 
of  the  process,  the  peculiarities  of  the  organism,  and  the  resistance 


INFECTIONS.  11 

of  body  tissues.  The  more  common  general  reactions  are  fever, 
digestive  disturbances,  effects  on  the  nervous  system,  leucocvtosis, 
anaemia,  and  enlargement  of  the  spleen  and  other  glands. 

Infections  and  infectious  diseases  may  be  divided  into  two 
classes ;  one  in  which  the  disease  runs  a  self -limited  course,  recovery 
coming  after  a  definite  period  of  time  has  elapsed,  while  in  the 
other  class  the  course  ma}"  be  extended  over  a  very  indefinite 
period  of  time. 

The  elimination  of  the  casual  or  etiological  factor  of  infections 
and  infectious  diseases  occurs  in  various  ways.  When  the  seat 
of  the  disease  is  on  the  surface  of  the  body,  the  organisms  are  elim- 
inated with  the  diseased  tissues,  but  when  the  organisms  are 
found  in  ]:arts  of  the  body  where  this  is  impossible,  the  microor- 
ganisms must  enter  the  general  circulation,  be  allowed  to  pass 
through  the  glandular  tissues  and  be  eliminated  with  the  secretions 
and  excretions.  In  the  blood  and  the  body  tissues,  bacteria 
are  in  many  cases  destroyed  by  bacteriolytic  and  bactericidal  sub- 
stances. Sometimes  after  an  apparent  recovery  from  the  disease, 
viable  organisms  may  remain  and  at  a  later  time  again  give  rise 
to  disease. 


CHAPTER  II. 


IMMUNITY 


Immunity  may  be  defined  as,  non-susceptibility  to  a  disease, 
or  as  the  ability  to  resist  the  action  of  the  causes  of  the  disease. 
The  body  may  be  immune  because  of  inherited  properties  or 
because  it  has  become  so  during  life.  Immunity  because  of  in- 
herited properties  is  called  "natural"  immunity,  while  the  mi- 
munity  acquired  during  life  is  called  "acquired"  immunity. 

Natural  immunity  is  demonstrated  by  the  non-susceptibility 
of  the  hen  to  the  action  of  the  tetanus  bacillus.  It  is  an  im- 
munity of  race  or  species.  This  immunity  at  times  may  be  re- 
duced or  removed  by  hunger,  exhaustion,  exposure  to  cold,  etc. 
Certain  closely  related  races  or  species  of  animals  sometimes  show 
a  natural  immunity  and  at  times  a  natural  susceptibility  to  the 
same  infecting  agent.  This  non-susceptibility  is  frequently  called 
a  natural  resistance  and  at  times  is  only  an  apparent  immunity, 
depending  in  these  cases  on  the  common  natural  barrier  to  the 
entrance  and  development  of  disease  producing  organisms.  xVgain, 
what  may  be  regarded  as  a  natural  immunity  is,  in  part  at  least, 
only  a  resistence  to  infection  due  to  the  inability  of  organisms  to 
reach  viable  tissues.  This  is  the  case  when  the  acidity  of  the 
stomach  is  sufficient  to  kill  cholera  organisms  before  they  reach 
the  epithelium  of  the  intestine. 

Acc[uired  immunity  only  results  after  a  pathological  condi- 
tion exists  or  has  existed.  The  individual  becomes  immune, 
because  he  has  survived  a  natural  course  of  the  disease,  as  is  the 
case  following  an  attack  of  scarlet  fever;  because  he  has  gone 
through  a  modified  form  of  the  disease,  as  is  the  case  in  vaccination 
against  small-pox;  or,  because  he  receives  substances  prepared  by 
some  other  individual  or  animal  tliat  has  gone  through  a  natural 


IMMUNITY.  13 

or  modified  course  of  the  disease.     Acquired  immunity  may  be 
either  active  or  passive. 

Experimental  active  immunization  is  usually  called  vacci- 
nation and  generally  produces  in  the  individual  a  modified  form 
of  the  disease.  All,  or  nearly  all,  of  the  symptoms  are  less  severe 
than  in  the  natural  course  of  the  disease.  The  individual  in  this 
case  produces  his  own  immunity.  In  artificial  or  intentional  in- 
oculation, the  etiological  factors,  or  more  particularly,  the  causal 
organisms  injected  must  be  so  modified  that  the  natural  course 
of  the  disease  will  not  follow  the  inoculation  or  injection.  Ex- 
perimentally acquired  active  immunity  is  produced  by  the  in- 
jection of  living  or  killed  microorganisms,  or  of  toxins  produced 
by  these  organisms.  When  living  organisms  are  injected  their 
virulence  is  usually  reduced  by  passage  through  animals,  growth 
at  high  temperatures,  on  artifical  media,  in  the  presence  of  chem- 
icals, or  growth  in  the  presence  or  absence  of  oxygen,  etc.  Active 
immunity  may  also  be  acquired  as  a  result  of  injection  of  living 
virulent  organisms  into  such  parts  of  the  body  where  the  disease 
will  not  be  produced.  The  first  amounts  injected  are  usually 
smaller  than  those  in  subsequent  injections.  When  bacteria  are 
injected  the  immunity  produced  is  a  bacterial  immunity  and 
when  toxins  are  injected  a  toxin  immunity  results. 

In  passively  acquired  immunity,  the  individual  that  becomes 
immune  does  little  or  nothing  toward  obtaining  this  immunity. 
It  results  from  the  injection  of  immunizing  substances  which 
have  been  prepared  by  an  actively  immunized  individual  or 
animal. 

There  are  two  classes  of  immunizing  substances;  those 
that  act  on  bacteria,  said  to  be  anti-bacterial;  and  those  that 
act  on  toxins,  called  anti-toxic.  Of  these  two  classes  of  immune 
substances,  the  anti -toxic  has  been  more  efficient  in  passive  im- 
munization. 

After  the  formation  of  immunizing  substances  they  do  not 
remain  indefinitely  within  the  body,  but  are  lost  through  the  ex- 
cretions either  as  immune  bodies  or  as  immtme  bodies  broken 
down  by  the  body  cells.  On  this  the  difference  in  persistance  of 
natural  and  acquired  immunity  is  partly  based,  for  in  acquired 
immunity  the  supply  of  immtme  bodies  is  exhausted,  while  in 
natural  immunity  new  substancss  are  constantly  formed. 


14  VACCIXE    AND    SERUM   THERAPY. 

THEORIES  OF  IMMUNITY. 


Acquired  immunity,  as  has  been  stated,  results  because  some 
individual  or  animal  has  gone  through  a  natural  or  modified 
course  of  the  disease.  While  acquiring  immunity,  the  body  and 
its  tissues  have  in  some  way  been  modified.  Various  theories 
have  been  advanced  to  explain  this  phenomenon. 

Klebs  and  Pasteur  tried  to  explain  the  changes  that  occur 
in  the  acquisition  of  immunity  by  assuming  that  in  going  through 
a  natural  or  modified  course  of  disease,  certain  substances,  nec- 
essarv  as  food  for  the  parasites,  are  used  up.  As  the  result  of 
immunization,  the  food  necessarv^  for  the  microorganism  is  con- 
sumed and  the  individual  is  immune  to  a  certain  organism  be- 
cause this  organism  cannot  obtain  the  food  it  needs  for  its  ex- 
istence and  production  of  the  disease. 

Chauveau  assumes  that  in  immunization  certain  products 
of  bacterial  metabolism  are  retained  in  the  body  of  the  immun- 
ized animal  or  individual,  which  products  protect  the  bod\'  tissues 
from  further  invasions  by  that  particular  parasite. 

Metchnikoft',  in  1883,  formulated  a  theory  according  to  which, 
during  immunization,  certain  white  blood  cells  and  cells  of  certain 
organs  acquire  the  ability  to  engulf  and  destroy  the  attenuated 
bacteria.  This  results  in  acquiring  the  abihty  to  engulf  and  de- 
stroy more  virulent  forms  of  this  same  species  of  microorganisms. 

EHRLICH'S    SIDE   CHAIN   THEORY   OF   IMMUNITY. 

In  1887  Salmon  and  Smith,  Foa  and  Bonome,  Roux  and 
Chamberland,  and  others  found  that  immunity  could  be  produced, 
not  only  bythe  injection  of  bacteria,  but  also,  as  a  result  of  the  injec- 
tion of  the  products  of  bacterial  metabolism.  As  a  result, a  chemi- 
cal theory  of  immunity  was  advanced.  According  to  this  theory 
the  tissues  of  the  body  are  chemically  changed  by   immunization. 

Fodor,  in  1887,  was  the  first  to  observe  that  the  normal  body 
fluids,  especially  the  blood,  contain  certain  substances  able  to 
destroy  bacteria.  Buchner,  Behring,  and  Nuttall,  soon  after 
Fodor's  observation  also  recognized  the  bactericidal  powers  of 
certain  sera.  In  1889  Buchner  reported  that  the  cell-free  blood 
serum  contains  certain  substances  which  he  called  alexines. 
Alexines,    he   found,    have   the   property   of   destroying  bacteria. 


EHRLICH  S   THEORY   OF   IMMUNITY.  15 

In  1888  Hericourt  and  Richet,  and  in  1889  Babes  and  Lepp  re- 
ported investigations  which  showed  that  by  injections  of  blood 
serum  from  animals  having  acquired  immunity  to  certain  diseases, 
immunity  to  these  same  diseases  can  be  conferred  to  other  ani- 
mals. Soon  after  this  Behring  and  Kitasato  reported  successful 
immunization  of  rats  to  tetanus  by  means  of  injections  of  blood 
serum  from  ral^bits  immunized  to  tetanus.  From  the  work  of  these 
investigators  there  developed  the  humeral  theory  of  immunity. 

Behring  and  Knorr  found  that  the  toxic  product  of  the  meta- 
bolism of  the  tetanus  bacillus,  could,  without  the  presence  of  the 
bacillus,  be  used  in  immunization.  The  serum  thus  produced,  they 
found  would  protect  against  the  disease  and  the  injection  of  the 
by-products  of  the  tetanus  bacillus.  They  assumed  from  this 
that  toxin  is  neutralized  by  the  immunizing  substance  in  the  .'m- 
mune  serum,  as  a  base  is  neutralized  by  an  acid. 

Ntunerous  theories  have  been  advanced  to  explain  the  facts 
observed  and  reported,  but  of  all  these  theories  one  stands  out 
prominently — Ehrlich's  side  chain  or  receptor'  theory,  advanced 
by  Ehrhch  in  1897. 

i\ccording  to  this  theory,  every  living  cell  consists  of  a  domi- 
nating nucleous  (Leistungskem)  and  of  side  chains  or  receptors. 
The  paradigm  of  this  picture  is  to  be  found  in  the  benzol  ring 
with  its  side  chains.  The  side  chains  or  receptors  of  a  cell  are  of 
many  different  kinds  and  serve  usually  to  anchor  and  assimilate 
the  food  stuffs.  At  times,  however,  by  means  of  the  receptors 
the  cells  are  combined  with  substances,  not  foods  but  cell  poisons. 
The  combination  of  the  receptors  of  the  cell  with  the  receptors  of 
foods  differs  from  the  coinbination  of  receptors  of  the  cell  and  re- 
ceptors of  toxins.  The  combination  of  the  receptors  of  the  cell 
with  the  receptors  of  foods  is  so  loose  that  after  undergoing  certain 
changes  which  are  of  value  in  assimilation  of  the  food  and  the 
nourishment  of  the  cell  the  food  stuff  is  again  eliminated  without 
having  injured  the  receptor.  Toxins  on  the  other  hand,  combine 
so  firmly  with  the  receptor  of  the  cell  that  they  cannot  again 
be  separated.  As  a  result  of  this,  receptors  anchoring  toxins 
are  lost  to  the  cell. 

When  a  certain  limit  of  anchoring  of  receptors  by  toxins  has 
taken  place  the  cell  peri  hes,  and  if  a  sufficient  number  of  cells 
which  have  vital  functions  to  perform  are  destroyed,  death  of 


16  VACCINE    AND    SERUM   THERAPY. 

the  individual  follows  When,  however,  the  toxins  do  not  anchor 
enough  receptors  or  are  not  potent  enough  to  destroy  the  cell 
and  the  organism,  immunity  results  or  follows. 

In  1896,  Weigert  advanced  the  hypothesis  that  when  fssue 
is  lost  the  regeneration  that  follows  is  not  only  sufficient  to  restore 
the  amount  of  tissue  lost,  but  actually  results  in  overproduction 
of  this  tissue.  This  process  is  observed  in  the  over-production 
of  segments  in  reptiles  and  of  cell  proliferation  in  granulating 
wounds  in  man  and  the  animals.  Ehrlich  assumes  that  when 
the  toxins  combine  with  the  receptors  of  the  cell,  the  receptors 
are  lost  to  the  cell  and  as  a  result  the  cell  is  stimulated  to  repro- 
duce the  destroyed  receptors.  The  process  does  not  stop,  how- 
ever, when  all  lost  receptors  have  been  reproduced,  for  more  re- 
ceptors will  be  produced  than  were  anchored  by  the  toxins.  The 
excessive  production  of  receptors  as  a  result  of  the  stimulus  fol- 
lowing the  anchorage  of  the  receptors  by  toxins,  Ehrlich  assumes, 
leads  to  a  disturbance  of  the  equilibrium  of  the  cell.  As  a  result 
of  'this,  the  surplus  receptors  are  thrown  off  and  constitute  what 
is  known  as  the  anti-body.  This  process  can  be  actually  ob- 
served in  the  lower  animals  in  which  extra  parts,  produced 
as  a  result  of  destruction  of  some  parts,  are  cast  off  or  lost. 

The  principal  function  of  all  receptors  and  side  chains  is  to 
provide  for  the  nutrition  and  metabolism  of  the  cells.  Receptors, 
and  hence  immune  bodies,  however,  are  not  all  of  the  same  com- 
position or  even  of  the  same  general  structure.  In  order  to  ex- 
plain the  different  functions  and  actions  of  different  immune 
bodies,  Ehrlich  has  assumed  that  the  receptors  may  be  of  simple 
constitution  and  structure  or  they  may  be  complex.  Any  cell 
of  the  body  may  have  large  numbers  of  the  same  and  different 
kinds  of  receptors.  Ehrlich  divides  this  large  number  of  receptors 
into  three  orders. 

RECEPTORS  OF  THE  FIRST  ORDER. 

These  are  receptors  that  are  of  relatively  simple  constitution 
and  structure  and  combine  with  substances  that  can  be  easily 
and  readily  anchored.  Bacterial  toxins  anchor  receptors  of 
this  order.  The  receptor  here  consists  of  only  one  haptophore, 
or  combining  group,  which  combines  directly  with  the  hapto- 
phore group  of  the  bacterial  toxin.     This  order  of  receptors  and 


RECEPTORS FIRST   AND    SECOND   ORDER. 


17 


immune  bodies  is  demonstrated  in  Figure  1,  and  represents  the 
type  of  receptor  on  which  is  based  the  action  of  bacterial  toxin 
and  the  formation  of  anti-toxin. 


/oxo-i'Hotfe.  Q-ifo</f 


X  OrfoetT 


~Fr^E.e.  "TfecsfTOT^, 


^ — ^o%iM  Tfaoor  To 


Fig.  1. 


RECEPTORS  OF  THE   SECOND  ORDER. 

The  receptors  of  the  second  order  are  distinguished  from 
those  of  the  first  order  in  that  the  receptors  here  have  in  addition 
to  the  haptophore  group,  a  zymophore  group.  This  zymophore 
group  acts  on  the  larger  food  particles,  making  them  more  readily 
assimilable.  In  a  similar  manner  it  acts  on  the  bacterial  cell. 
Receptors  of  this  kind,  possessing  haptophore  and  zymophore 
groups,  are  broken  off  from  the  cells  and  circulate  in  the  blood  as 
agglutinins  and  precipitins  after  immunization  by  the  injection 
of  certain  bacteria.  The  haptophore  group  of  the  immune  body 
in  an  agglutinating  or  precipitating  serum  combines  with  the 
bacterial  cells.  The  zymophore  group,  however,  does  not  com- 
bine with  anything  but  exerts  its  influences  entirely  through  the 
haptophore  group.  The  zymophore  group  is  destroyed  by  age, 
acids,  heating  to  65°  C,  etc.  Receptors  and  immune  bodies  of 
the  second  order  are  represented  in  Figure  2. 


jB^crs 


•21  y/«^07'«<J7f e   (^ffOUP 


Fig.  2. 


18 


VACCINE    AND    SERUM   THERAPY 


RECEPTORS  OF  THE  THIRD  ORDER 
The  receptors  of  the  third  order  are  Hkewise  adapted  to  the 
anchorage  of  bacteria.  These  receptors,  however,  have  two  com- 
bining groups:  one  for  the  anchorage  of  cells  or  food  substances 
and  the  other  for  the  anchorage  of  substances  which,  acting  through 
the  receptors,  can  produce  a  ferment-like  action.  This  latter 
substance  is  called  an  activating  substance  and  is  present  in  nor- 
mal sera.     It  is  known  either  as  complement  or  alexin. 

Complement  is  easily  destroyed  by  ageing,  acids,  heat,  55°  C, 
being  sufficient  to  destroy  it.  When  the  receptors  of  this  order 
are  anchored  by  bacteria  in  numbers  not  sufficient  to  kill  the  cell 
but  to  stimulate  it  to  over-production  of  receptors,  the  extra 
receptors  are  thrown  off.  The  receptors  thrown  off  constitute 
the  immune  body,  amboceptor  or  substance  sensibilitrice  of  Bor- 
det.  They  have  two  combining  or  haptophore  groups,  one  for 
the  combination  with  bacterial  or  other  cells  and  substances, 
called  the  "cytophyUc"  group;  the  other  for  combination  with 
alexin  or  complement,  called  the  " complement ophore"  group. 
It  is  by  means  of  complement  that  the  amboceptor  is  able  to 
dissolve  bacteria,  red  blood  cells  and  other  substances.  Immune 
sera  containing  receptors  of  the  third  order  are  bacteri- 
olytic, haemolytic,  or  cytol3rtic,  depending  upon  wdiether  they, 
together  with  complement,  can  dissolve  bacteria,  red  blood  cells, 
or  other  cells.  The  receptors  of  the  third  order  are  graphically 
represented  in  Figure  3. 


Tfec-p-rntf  J2L  OffDETf 


(^yrOTHf  UlC    (f-rfOi/P, 


Fig.  3. 


It  will  be  observed  that  receptors  of  the  first,  second  or  tliird 
order  can  be  produced  in  excess  to  form  immune  body  or  anti- 


ANTI-BODIES.  U) 

body,  specific  immune  bodies  are  produced  for  many  substances, 
toxins,  bacterial  cells,  red  blood  cells,  ferments,  etc.  As  a  result 
of  this  multiplicity  of  stibstances  that  can  cause  the  cells  to 
produce  anti -bodies,  anti- bodies  ma)^  be  anti- toxic,  aggluti- 
nating, precipitating,  lytic,  etc.  To  produce  agglutination, 
precipitation  or  lysis  it  is  necessary  that  a  ferment-like  sub- 
stance be  a  part  of,  or  able  to  combine  with,  the  immune  body. 
This  ferment -like  substance,  as  stated  before,  is  labile  and  lost 
on  ageing,  heating,  etc.  When  this  is  lost  the  serum  containing 
the  immune  body  is  said  to  be  inactive.  In  normal  serum  there 
is  present  a  substance  called  complement  which  can  again  reacti- 
vate a  lytic  serum.  The  agglutinating  power,  however,  cannot 
be  restored  by  the  addition  of  fresh  complement.  Complement 
can  be  preserved  for  long  periods  of  time  if  the  serum  is  dried 
without  heat  soon  after  the  blood  is  drawn.  Whether  there  is 
only  one  complement  or  a  multiplicity  of  the  same  has  not  been 
definitely  decided,  Ehrlich  assuming  that  there  is  a  multiplicity 
of  the  same,  while  Bordet  and  Buchner  claim  there  is  only  one. 

It  has  been  demonstrated  that  anti-toxic  substances  are  im- 
portant in  preventing  the  action  of  toxins  on  the  body  cells,  while 
lytic  substances  have  been  found  to  protect  the  body  by  the 
solution  and  destruction  of  bacteria.  Whether  the  agglutinat- 
ing or  precipitating  substances  are  of  importance  in  destroying 
bacteria,  is  by  no  means  certain.  Numerous  investigators  as- 
sume that  they  injure  and  change  the  bacterial  cells  or  substances 
used  in  immunization,  while  others  claim  that  they  exert  no 
such  action.  It  has  been  quite  definitely  observed  that  it  is  pos- 
sible to  cultivate  and  grow  bacteria  that  have  undergone  ag- 
glutination. 

As  has  been  stated  earlier,  immune  bodies  are  formed  only 
for  such  substances  as  are  able  to  combine  firmly  w^ith  the  recep- 
tors of  the  cells,  and  it  is  on  this  assumption,  that  Ehrlich  explains 
the  impossibility  of  producing  immunity  to  certain  poisons,  as 
the  alkaloids,  strychnine  and  morphine. 

The  cells  whose  receptors  anchor  the  substances  and  cells  for 
which  immune  substances  are  formed,  are  probably  widely  dis- 
tributed throughout  the  body.  The  particular  tissues  in  which 
anti-bodies  are  formed  has  not  been  determined  and  probably 
varies  for  the  difi'erent  substances  to  which  immunization  can  be 


20  VACCIXI-:    AXD    SKRl'M    THI-:RAPy. 

obtained.  It  seems  quite  definite  that  in  certain  cases,  internal 
organs  may  contain  more  immune  substances  than  the  blood  se- 
rum. After  immune  bodies  have  heen  formed  they  do  not  remain 
permanently  in  the  body,  but  are  gradually  lost  through  destruc- 
tion in  the  body  or  lost  with  the  excretions. 

Ehrlich's  views  on  the  formation  of  immune  bodies  are  cjuite 
generally  accepted.  His  views,  however,  concerning  the  com- 
bination of  toxin  and  anti -toxin,  agglutinable  substances  and 
agglutinins,  and  substances  capable  of  solution  and  lysins  are 
not  as  universally  accepted.  Ehrlich  assumes  that  the  union  of 
immune  bodies  and  the  specific  substances  is  a  chemical  combina- 
tion. Bordet  assumes  that  in  lytic  sera  the  immune  body  acts 
as  a  mordant  which  sensitizes  the  substance  capable  of  solution 
to  the  action  of  the  complement.  Field  has  explained  the  inter- 
action of  immune  bodies  and  specific  substance  according  to  the 
principles  of  colloid  chemistry,  so  that  for  example  in  a  toxin 
and  anti-toxin  combination  one  is  adsorbed  (physical  rather  than 
chemical)  by  the  other. 

OPSONIN  THEORY  OF  IMMUNITY. 

It  has  been  stated  earlier  that  in  1883,  Metchnikoff  advanced 
his  phagocytic  theory  of  immunity.  According  to  this  theory 
the  presence  or  absence  of  immunity  depends  upon  the  ability  of 
phagocytes  to  engvilf  and  destroy  bacteria.  Metchnikoff  had 
given  serum  little  consideration  in  immunity  although  he  believed 
that  some  sera  contain  substances  which  stimulate  the  leucocytes 
to  engulf  bacteria.  These  substances  he  called  "stimulins."  The 
phagocytic  theory,  however,  was  practically  replaced  by  the 
humeral  theory  of  immunity  because  Fudor,  Buchner,  Behring, 
Nuttall  and  others  had  found  that  serum  containing  no  cells 
whatever  can  destroy  bacteria  by  solution.  It  was  also  found 
that  antitoxin,  which  combines  with  toxin,  is  carried  in  serum. 

From  1887  on  the  humeral  theory  of  immunity  gained  a 
strong  foothold  and  it  was  not  until  1895  that  attention  was  again 
called  to  tlie  function  of  the  leucocytes  in  immunity.  In  this 
year  Denys  and  LeClef  reported  experiments  from  which  they 
concluded  that  in  immunization  certian  changes  are  produced 
in  the  serum,  which  make  it  possible  for  the  leucoc}'i:es  to  engulf 
bacteria.     In  their  conclusions  they  state  that  leucocytes  from  a 


OPSONIN    THEORY    OF    IMMUNITY.  21 

normal  rabbit  on  the  addition  of  normal  rabbit  serum,  have  only 
slight  phagocytic  action  for  the  streptococcus.  If,  however, 
leucocytes  from  a  normal  rabbit,  or  from  a  streptococcus-immune 
rabbit  are  added  to  the  serum  from  an  animal  vaccinated 
with  streptococci,  the  leucocytes  actively  engulf  and  destroy 
the  streptococci.  When  streptococci,  able  to  produce  erysipelas 
in  a  normal  rabbit,  are  injected  under  the  skin  of  a  rabbit  vacci- 
nated against  streptococci,  disease  is  not  produced.  This  pro- 
tection is  due,  according  to  these  investigators,  especially  to  the 
ability  of  the  leucocytes  to  destroy  streptococci.  They  attribute 
the  increased  phagocytosis  to  an  action  of  the  immune  serum  on 
the  leucocytes. 

The  results  obtained  and  conclusions  arrived  at  by  Denys 
and  LeClef  were  repeated  by  Bordet,  who  was  not  able  to  verify 
the  results  obtained  by  these  investigators.  Mennes  found  that 
immunity  to  Mic.  pneumoniae  depends  on  the  action  of  serum 
which  produces  active  phagocytosis  but  was  not  certain  that 
this  action  is  due  to  stimulation  of  the  leucocytes. 

In  1903  Wright  and  Douglas  pointed  out  that  there  are  cer- 
tain substances  in  serum  that  so  affect  bacteria  that  they  are 
more  easily  taken  up  and  disposed  of  by  the  leucocytes.  This 
substance  they  called  "opsonin."  They  found  opsonin  present  in 
normal  and  immune  serum  and  from  their  investigations  decided 
that  the  amount  present  in  serum  is  variable,  and  can  be  increased 
or  decreased  by  the  injection  of  killed  cultures  of  bacteria.  To 
determine  the  amount  of  opsonin  present  in  blood  they  modified 
the  method  of  estimating  phagocytic  power  introduced  by  Leish- 
man  in  1902.  According  to  their  method  the  average  number 
of  bacteria  taken  up  by  leucocytes,  when  bacteria,  leucocytes  and 
serum  are  mixed  together  and  allowed  to  remain  together  for  a 
certain  time,  is  determined.  The  ratio  of  the  average  number 
of  bacteria  per  leucocyte  when  patient's  seruin  and  serum  from 
a  healthy  individual  determine  the  phagocytosis,  they  called  the 
opsonic  index.  The  methods  employed  in  the  determination  of 
this  opsonic  index  will  be  taken  up  later. 

In  1904  Neufeld  and  Rimpau,  entirely  independently  of 
Wright  and  Douglas  referred  to  the  two  well-known  elements, 
antitoxin  and  bactericidal  substances,  in  immune  serum  and  stated 
that  in  anti -streptococcic  and  anti-pneumococcic  serum  they  had 


22  VACCINE    AND    SERUM    THERAPY. 

found  a  third  factor  of  importance  in  immunity.  This  element, 
accordmg  to  these  investigators,  sensitizes  the  corresponding 
bacteria  and  so  modifies  theva  that  they  are  more  readily  engulfed 
by  the  leucocytes.  This  third  factor  in  immunity  they  called 
"bacteriotropin."  It  is  to  be  noticed  that  Neufeld  and  Rimpau 
worked  with  virulent  streptococci  and  pneumococci.  These  or- 
ganisms are  not  ingested  by  the  leucocytes  in  a  mixture  of  leuco- 
cytes, virulent  streptococci  or  pneumococci  and  normal  serum. 
However,  when,  instead  of  normal  serum,  a  specific  anti-serum 
takes  part  in  the  mixture  there  is  ingestion  even  of  cultures  of 
virulent  strains  of  these  organisms.  They  furthermore  showed 
that  the  serum  does  not  act  on  the  leucocytes  but  exerts  its  in- 
fluence on  the  bacteria,  sensitizing  them  to  the  action  of  leuco- 
cytes. 

There  seems  now  to  be  but  little  doubt  that  opsonin  and 
bacteriotropin  are  the  same  substances,  the  greatest  difterence 
that  can  be  assigned,  being  that  bacteriotropins  are  probably 
what  Wright  and  Douglas  have  called  "immune  opsonins." 


Most  investigators  now  take  an  intermediate  position  on  the 
phagocytic  and  humeral  theories  of  antibacterial  immunity.  It  is 
quite  generally  accepted  that  both  serum  and  leucoc\^es  contain 
substances,  which,  acting  after  the  manner  of  ferments,  are  able 
to  dissolve  bacteria.  Just  as  all  important  manifestations  of  life 
are  found  in  the  normal  and  pathological  cellular  elements,  so  also 
the  means  of  defense  against  harmful  agents  is  probably  closely 
related  to  the  condition  and  functions  of  cells  which  prepare  and 
secrete  the  protective  substances  by  means  of  which  bacteria  and 
other  harmful  agents  are  destroyed  or  neutralized. 


CHAPTER  III. 


THE  OPSONIC  INDEX 


From  the  foregoing  chapter  it  is  seen  that  since  the  work 
of  Denys  and  LeClef  in  1895,  it  has  been  known  that  serum  is  of 
importance  in  phagoc3rtosis.  Little  attention,  however,  was 
given  to  the  various  observations  on  this  subject  until  the  work 
of  Wright  and  Douglas  was  presented.  Although  Neufeld  and 
Rimpau  discovered  probably  the  same  substances  as  Wright  and 
Douglas,  these  investigators  are  seldom  referred  to  in  the  discusion 
of  the  substances  that  change  bacteria  so  as  to  prepare  them  tor 
ingestion  by  leucocytes.  There  are  two  definite  scientific  reasons 
for  the  great  prominence  given  to  the  work  of  Wright  and  Doug- 
las: one  is  that  they  advanced  and  improved  a  technique  for  the 
determination  of  the  amount  of  phagocytosis,  by  means  of  which 
the  opsonic  index  could  be  determined;  the  other  is  to  be  found 
in  the  widespread  interest  in  the  methods  they  advanced  for 
active  immunization  of  patients  by  the  injection  of  killed  cultures. 

In  1902  Leishman  presented  a  method  for  the  quantitative 
determination  of  phagocytosis.  He  mixed  equal  quantities  of 
patient's  blood  and  bacterial  emulsion,  which  he  then  incubated 
for  a  time  in  a  moist  chamber.  After  incubation  he  made  cover 
glass  spreads  which  he  dried,  fixed  and  stained.  On  these  slides 
he  counted  the  number  of  bacteria  ingested  by  the  leucocytes, 
from  which  the  average  number  per  leucocyte  was  determined. 
This  average  he  compared  with  the  average  per  leucocyte  obtained 
when  normal  blood  instead  of  patient's  was  added  to  the  bacterial 
emulsion.  Leishman  did  not  take  into  consideration  the  action 
of  serum  on  bacteria  or  leucocytes  but  devised  merely  a  "method 
of  estimating  phagocytic  power." 

Wright  and  Douglas,  in  their  work  on  the  determination  of 
the  opsonic  index  modified  Leishman 's  method  to  meet  their 
theory  on  phagocytosis.     According  to  their  observation  opsonin 


24  VACCINE   AND    SERL'M   THERAPY. 

is  a  substance  present  in  all  serum.  The  amount  varies  in  the 
different  sera  and  as  the  amount  varies  so  also  will  the  amount  of 
phagocytosis  vary.  To  determine  the  phagocytic  power  it  is 
necessary  to  have  the  three  factors  of  phagocytosis  in  the  mix- 
ture. These  three  factors  are  found  in  the  blood  serum,  leuco- 
cytes and  bacteria.  A  mixture  of  these  three  must  be  allowed 
to  remain  together  for  a  definite  period  of  time,  after  which  the 
average  number  of  bacteria  taken  up  by  the  polymorphonuclear 
neutrophiles  must  be  determined.  This  average  number  of  bac- 
teria per  leucocyte  is  called  the  "phagocytic  index."  The 
phagocytic  index  obtained  when  serum  from  the  patient  is  mixed 
and  incubated  with  leucocytes  and  bacteria,  is  compared  with 
the  phagocytic  index  obtained  when  serum  from  a  healthy  in- 
dividual is  added  to  and  incubated  with  a  similar  amount  of  the 
same  emulsions  of  bacteria  and  leucocytes.  The  result  obtained 
is  called  the  "opsonic  index." 

The  technique  here  given  is  essentially  the  one  developed 
by  Wright  and  Douglas  and  demonstrated  in  New  York  City  in 
1906  by  Wright  and  taught  by  his  associate,  Dr.  Ross.  It  is 
given  somewhat  in  detail  inasmuch  as  it  is  the  one  most  generally 
used,  even  though  many  modifications  have  been  suggested  and 
followed  by  different  investigators. 

SERUM.  . 
Blood  from  the  individual  whose  serum  is  to  be  used  in  the 
determinations  of  the  opsonic  index,  is  collected  as  follows:  A 
small  glass  capsule  with  one  curved  capillary  limb  is  made  as  is 
indicated  in  Fig.  4.  This  tube  is  brought  to  a  needle's  point  at 
"b"  by  heating  over  the  pilot  flame  of  a  Bunsen  burner.     This 


Fig.  4. 

point  is  later  used  to  puncture  the  finger  or  lobe  of  the  ear.     The 
end  "a"  of  the  capsule  is  left  open.     One  of  the  fingers  of  the 


COLLECTION    OF    SERUM. 


jLO 


hand  is  cleaned  with  alcohol  and  water,  after  which  a  bandage, 
handkerchief  or  rubber  band  is  quite  firmly  tied  around  this  fin- 
ger. The  windings  of  the  bandage,  handkerchief  or  rubber  band 
are  started  at  the  base  of  the  finger  and  run  gradually  toward 
the  tip  of  the  finger  as  more  turns  are  put  on.  In  this  way  there 
is  produced  an  accummulation  of  blood  in  the  veins  and  capil- 
laries. Now,  with  the  pointed  end  of  the  capillary  bulb,  the  fin- 
ger is  pricked  at  a  point  about  a  quarter  of  an  inch  back  of  the 
nail,  the  needle  end  is  broken  off  enough  to  open  the  capillary 
end.  The  open  end  "a"  is  now  held  close  to  the  drop  of  blood 
which    gradually    fills    the    bulb;    this    is    indicated    in    Fig.    5. 


Fig.  5. 

When  enough  blood  has  been  drawn  and  collected  in  the  bulb,  the 
bulb  is  held  between  the  fingers  at  "a"  and  "c"  and  the  end"b"  is 
heated  and  sealed  off.  As  the  end  "b"  cools  the  blood  will  draw 
away  from  the  end  "a"  and  finally  all  the  blood  will  collect  in 
the  bulb  proper.  After  this  the  bulb  is  again  taken  hold  of  at 
the  curve  "c"  and  by  a  rapid  swing  of  the  arm,  as  is  practiced 
in  shaking  down  a  clinical  thermometer,  the  blood  is  thrown  down 
into  the  sealed  end  "b"  as  is  indicated  in  Fig.  6.  The  blood  is 
now  allowed  to  clot  in  order  to  expel  the  serum.  The  serum 
may  also  be  separated  by  hanging  the  bulb  at  "c"  over  the  arm 
of  the  centrifuge,  and  centrifuging  until  the  clot  and  serum  have 


26  VACCINE    AND    SERUM    THERAPY. 

well  separated.     The  tube  may  now  be  opened  by  filing  and  break - 
incr  at  "c." 


Fig.  6. 

According  to  the  original  method  of  Wright  and  Douglas, 
blood  was  obtained  from  the  patient  and  from  a  number  of 
healthy  individuals.  Equal  parts  of  the  normal  serum  were  taken 
and  mixed  together  or  "pooled."  This  was  done  so  as  to  get  a 
better  normal  serum.  After  it  has  once  been  established  that 
serum  from  a  certain  healthy  individual  has  an  opsonic  index 
of  1 . 0  for  a  particular  organism  when  compared  with  pooled 
serum,  this  individual's  serum  replaces  the  pooled  serum.  After 
this  has  been  established,  the  opsonic  index  of  any  patient  for 
this  organism  is  determined  by  dividing  his  phagocytic  index 
by  the  phagocytic  index  of  the  "normal  individual,"  as  he  may 
now  be  called. 

LEUCOCYTES. 

The  leucocytes  used  in  the  determination  of  the  opsonic 
index  are  usually  obtained  from  the  blood  of  supposedly  healthy 
individuals,  most  frequently  from  the  blood  of  the  investigator 
himself.  No  particular  stress,  however,  is  laid  upon  the  individ- 
ual from  whom  the  blood  is  obtained. 

To  obtain  blood,  preferably,  the  middle  finger  is  cleaned,  con- 
gested and  punctured  in  the  same  manner  as  was  followed  in  the 
collection  of  serum.  Ten  drops  of  blood  are  collected  in  about 
lOc.c.  of  a  normal  salt  solution  containing  one  per  cent  of  sodium 
citrate.  The  sodium  citrate  is  added  to  keep  the  blood  from 
clotting.  The  tube  containing  the  mixture  is  then  centrifuged 
in  a  centrifuge  of  not  too  great  speed.  When  the  speed  exceeds 
1,200  to  1,500  revolutions  per  minute,  the  cells  are  too  closely 


LEUCOCYTES    EMULSION.  27 

packed  together  and  form  clumps.  The  mixture  is  centrifuged 
until  the  corpuscles  are  thrown  to  the  bottom  of  the  tube  and 
the  fluid  above  is  clear,  although  it  may  be  slightly  straw 
colored.  The  supernatent  fluid  is  then  drawn  off  with  a 
pipette.  The  corpuscles  are  washed  free  from  serum  and  sodium 
citrate  by  again  filling  the  tube  with  normal  salt  solution,  mix- 
ing thoroughly  and  centrifuging  until  there  again  is  a  clear  super- 
natent fluid.  If  the  material  in  the  centrifuge  tube  is  now  ex- 
amined, one  sees  a  clear  fluid  above  and  a  red  fluid  in  the  lower 
part  of  the  tube.  The  red  fluid  below,  however,  is  not  of  the 
same  shade  throughout.  The  uppermost  part  consists  of  a  gray- 
ish red  layer,  the  leucocytes.  These  are  layered  above  the  red 
blood  cells  because  of  the  difference  in  specific  gravity  of  the 
leucoc5rtes  and  red  blood  cells.  It  is  from  the  gray  layer  that 
the  leucocytes,  to  be  used  in  the  determination  of  the  opsonic 
index,  are  obtained. 

To  obtain  the  leucocytes  the  clear  fluid  above  is  drawn  oft' 
with  a  pipette  and  then  with  a  clean  pipette  of  about  1 .  6m. m. 
inside  diameter,  the  leucocytes  are  removed.  To  do  this  the 
pipette  firmly  held  in  the  hand,  all  of  the  air  in  the  nipple 
is  expelled,  and  then  as  the  pressure  on  the  nipple  is  gradually 
released,  the  open  end  of  the  pipette  is  held  on  the  surface  of  the 
uppermost  layer  of  grayish  red  color.  This  fluid  will  contain 
many  red  blood  cells  and  also  a  relatively  large  number  of  leuco- 
cytes. After  this  fluid  has  been  drawn  into  the  larger  part  of 
the  pipette  the  capillary  end  is  sealed  off.  This  fluid  is  called 
"leucoc}'i:ic  cream."  Wright  and  Douglas  state  that  in  their 
experience  there  is  no  variation  of  the  ability  to  engulf  bacteria 
within  the  space  of  a  few  hours  but  that  after  three  days  the 
phagocytic  power  decreases  to  one-half  or  one-third  of  what  it 
was  when  freshly  drawn. 

BACTERIAL  EMULSION. 

Inasmuch  as  Wright  had  early  decided  that  opsonins  are 
specific,  such  species  or  varities  of  bacteria  are  used  in  the  deter- 
mination of  the  opsonic  index  as  may  be  of  importance  in  the 
bacterial  infection.  In  the  selection  of  the  particular  culture  to 
be  used,  there  are  two  sources— either  the  different  cultures  iso- 
lated from  the  lesion  are  used  or  else  the  same  species  and  varieties 


28 


VACCINE    AXD    SERUM    THERAPY. 


of  microorganisms  are  taken  from  a  stock  culture.  Usually  liv- 
ing freshly  grown  bacteria  are  used  to  make  emulsions.  The 
principle,  or  practically  only  exception  to  this  has  been  in  the 
determination  of  the  opsonic  index  for  the  bacillus  of  tubercu- 
losis, for  which  often  killed  and  old  cultures  of  the  organism  have 
been-  used  to  make  the  bacterial  emulsion. 

In  all  cases  it  is  intended  that  the  bacteria  shall  be  well  sep- 
arated and  suspended  uniformly  in  salt  solution.  The  tech- 
nique for  making  the  bacterial  suspension  varies  with  the  differ- 
ent organisms,  some  of  which  it  will  be  necessary  to  consider 
separately.  The  organisms  for  which  the  opsonic  index  is  de- 
termined may  be  divided  into  three  classes. 

A.  Many  of  the  organisms  belonging  to  this 
class  grow  on  the  ordinary  media,  while  for 
others,  media  containing  blood  or  other  body 
fluids  are  necessary.  The  organisms  occur 
singly  or  in  pairs  and  when  present  in  larger 
groups  the  cell  aggregates  can  be  easily 
broken  up. 

(a).  Mic.  pyogenes  aureus,  albus  and  citreus, 
B.  coli,  B.  typhosus,  B.  pyocyaneus,  etc.,  are 
grown  on  slant  agar  for  twenty-four  hours 
at  37°  C.  Various  investigators,  including 
Wright,  have  used  iour  to  five  hour  cultures 
of  these  organisms. 

(b).  Mic.  gonorrhoeas  has  usually  been 
grown  on  hydrocele  or  human  blood  agar. 
Cole  and  Meakins  obtained  their  cultures  from 
growth  on  agar  in  which  0.5  c.c.  of  fresh 
blood  had  been  added  to  10  c.c.  of  ordinary 
agar.  The  age  of  the  cultures  used  has  varied 
from  four  to  twenty-four  hours  incubation 
at  37°  C. 

(c).    Mic.   meninigitidis,    Mic.     pneumoniae, 
etc.,  produce   good   growth    on  sheep    serum 
agar.    The  cultures  have  usually  been  incubated  at  37°  C.  from 
twelve  to  twenty-four  hours. 

To  each  culture  of  the  proper  age  abotit  ten  drops  of  sterile 
normal  salt  solution  are  added.     With  a  platinum  loop  the  cul- 


FiG.  7. 


BACTERIAL    EMULSION.  29 

ture  is  washed  off  the  media  and  with  a  thick  walled  capillary 
pipette,  with  the  end  broken  off  squarely,  the  suspension  is  drawn 
into  the  pipette.  The  open  end  of  the  pipette  is  firmly  pressed 
against  a  watch  glass,  as  indicated  in  Fig.  7.  When  this  is  done 
only  a  small  crevice  will  remain  between  the  end  of  the  pipette 
and  the  watch  glass.  The  bulb  is  now  compressed  and  the  bac- 
teria suspended  in  the  salt  solution  are  forced  out  through  the 
small  crevices.  This  is  done  to  break  up  the  small  clumps  of  bac- 
teria. 

B.  The  organisms  belonging  to  this  class  grow  on  the  same 
kind  of  media  as  do  those  belonging  to  the  first  group.  The  or- 
ganisms of  this  group  form  cell  aggregates  that  are  not  easily 
broken  up.  The  streptococci  are  the  most  important  organisms 
in  this  class.  Streptococci  frequently  grow  in  long  chains, 
varying  from  two  to  thirty  cocci.  It  is  evident  that  a  leuco- 
cyte may  be  able  to  engulf  one  or  more  cocci  without  being  able 
to  engulf  a  chain  consisting  of  twenty  or  thirty  cocci.  The  method 
for  breaking  up  ordinary  clumps  will  not  suffice  for  breaking  up 
chains  of  streptococci. 

While  observing  the  opsonic  index  for  streptococci  in  ery- 
sipelas, the  writer  found  it  necessary  to  adopt  some  method  to 
break  up  the  long  chains  of  streptococci  frequently  isolated  from 
the  lesions  in  erysipelas.  This  method  consisted  in  the  addition 
of  2  to  3  c.c.  of  sterile  salt  solution  to  each  twenty-four  hour  cul- 
ture on  glycerine,  glucose  agar.  After  washing  off  the  growth, 
the  emulsion  was  put  into  a  small  test  tube  containing  sterile 
sea  sand;  the  tube  was  sealed  in  a  flame  and  shaken  for  one  and 
one-half  hours  in  the  shaking  machine.  The  sand  and  emulsion 
in  the  tube  were  centrifuged  for  one  minute  and  the  supernatent 
fluid  drawn  off.  In  this  way  short  chains,  from  two  to  four 
cocci,  were  obtained. 

Another  method  was  devised  by  Wright  of  Harvard  University. 
This  method  differs  only  from  the  one  of  Wright  and  Douglas  in  the 
substitution  of  a  paraffine  block  for  the  watch  glass.  When  the 
open  end  of  the  pipette  is  held  against  the  paraffine  the  crevices 
very  small  so  that  the  chains  are  more  completely  broken  up. 
This  method  gives  very  satisfactory  results.    . 

C.  This  class  includes  practically  only  one  species  of  mi- 
croorganisms,  the  bacillus  of  tuberculosis.     The  preparation   of 


30  VACCINE    AND    SERUM    THERAPY. 

suspensions  of  tubercle  bacilli  is  attended  by  numerous  difficul- 
ties, because  this  organism  when  grown  on  artificial  media  forms 
conglomerated  masses.  According  to  Wright  and  Douglas  the 
living  tubercle  bacilli  are  heated  to  100°  C.  before  breaking  up 
the  clumps.  Later  Wright  modified  this  techinque  by  heating 
the  bacilli  to  100°  C.  on  three  successive  days.  The  clumps  after 
this  are  ground  up  in  an  agate  mortar  or  in  a  watch  glass,  two  or 
three  drops  of  0. 1  per  cent  salt  solution  being  added  at  a  time  until 
two  or  three  c.  c.  have  been  added.  One-tenth  per  cent  salt 
solution  is  used  in  making  this  suspension  because  with  greater 
concentration  the  bacilli  are  again  clumped.  Later  Wright  added 
1 . 5  per  cent  salt  solution  in  maknig  up  the  emulsion  of  tubercle 
bacilli  because  he  found  that  this  concentration  is  necessary  to 
prevent  spontaneous  phagocytosis.  When  the  heated  bacilli  are 
thoroughly  rubbed  and  suspended  in  1 . 5  per  cent  salt  solution 
a  homogeneous  mixture,  containing  but  few  clumps  and  many 
isloated  bacteria,  results.  This  mixture  is  then  centrifuged  at 
high  speed  for  about  ten  minutes.  After  this  the  supernatent 
fluid  is  drawn  off,  and  enough  salt  sokition  added  to  get  the  right 
concentration  of  the  emulsion. 

While  this  method  gives  a  fairly  homogenous  suspension  of 
tubercle  bacilli,  still  in  an  emulsion  made  in  this  way  many  of 
the  tubercle  bacilli  are  broken  up.  In  determining  the  opsonic 
index  it  is  necessary  to  count  the  number  of  bacilli  taken  up  by 
the  leucocytes,  and  when  there  is  fragmentation  of  bacilli  it  is 
necessary  either  to  count  each  fragment  as  one  bacillus,  or  else  to 
determine  the  fractional  part  of  a  bacillus.  Either  of  these 
methods  is  most  unsatisfactory. 

Sellard  and  Jeans  have  emulsified  the  living  tubercle  bacilli 
in  the  same  manner  that  has  been  used  for  the  emulsification  of 
other  bacteria.  After  this  they  have  killed  the  bacilli  by  exposing 
the  emulsion  to  sunlight  for  a  number  of  hours — ten  hours  being 
sufficient  to  kill  all  tubercle  bacilli  present.  In  such  emulsions 
they  have  gotten  no  spontaneous  clumping,  no  fragmentation,  nor 
spontaneous  phagocytosis. 

Walker  has  recommended  a  method,  according  to  which  Dor- 
sett's  egg  medium  is  heavily  inoculated  with  an  actively  growing 
culture  of  the  bacillus  of  tuberculosis.  After  fourteen  to  eighteen 
hours  of  incubation  at  37°  C,  salt  solution  is  squirted  over  the 


MIXTURE    OF    BACTERIA,    LEUCOCYTES    AND    SERUM.  31 

culture,  which  is  now  rubbed  off  into  the  salt  solution,  the  clumps 
being  broken  up  with  a  platinum  needle.  The  suspension  is  then 
filtered  first  through  a  loosely  packed  cotton  filter  and  later  through 
a  filter  made  with  scraped  filter  paper.  Filtration  is  repeated 
until  all  clumps  are  removed,  after  which  the  clump-free  suspen- 
sion is  heated  to  75°  C.  for  twenty  to  thirty  minutes. 

STRENGTH  OF  BACTERIAL  EMULSION. 

The  strength  of  bacterial  emulsion  most  commonly  used  has 
a  slightly  opalescent  appearance.  It  has  been  found  that  cloudi- 
ness may  be  absent  or  be  only  very  slight  and  still  the  emulsion 
ma}^  contain  too  many  bacteria.  According  to  Wright's  instruc- 
tions the  strength  of  the  emulsion  to  be  used  is  one  which  gives 
an  average  count  of  from  five  to  eight  bacteria  for  leucocytes 
when  normal  serum,  leucocytes  and  the  suspension  of  bacteria 
are  incubated  for  the  proper  length  of  time.  Walker  has  obtained 
better  results  with  heavier  suspensions  of  bacteria  and  diluted 
serum.  It  is  not  very  difficult  to  make  an  emulsion  of  staphy- 
lococci, for  after  a  bit  of  experience  it  can  be  determined  by  the 
naked  eye  whether  the  suspension  of  this  species  is  heavy  enough 
or  not.  The  tubercle  bacillus,  however,  presents  greater  diffi- 
culties, it  frequently  being  necessary  to  actually  make  a  trial 
test  for  the  bacterial  emulsion.  Simon  and  others  have  proposed 
the  numerical  determination  of  the  number  of  bacteria  per  c.  c. 
in  the  bacterial  emulsion. 

MIXTURE  OF  BACTERIA,  LEUCOCYTES  AND  SERUM. 

From  the  definition  of  the  opsonic  index,  it  is  evident  that 
similar  quantities  of  the  same  factors  must  be  taken  to  make  com- 
parative mixtures.  Furthermore,  according  to  the  methods  de- 
vised by  Wright,  equal  amounts  of  each  factor  are  mixed  to- 
gether. When  it  is  desired  to  use  diluted  serum  equal  volumes 
may  still  be  used  if  the  serum  be  diluted  properly  before  taking 
the  volume. 

In  order  to  get  equal  volumes  Wright  has  made  a  capillary 
pipette,  the  walls  of  the  capillary  part  of  which  are  thick.  The 
end  of  the  pipette  is  broken  oft'  squarely.  About  a  quarter  or 
one-half  inch  from  the  open  capillary  end  a  mark  is  made  with  a 
soft  wax  pencil.     This  pipette  is  shown  in  Fig.  8.     By  means  of 


32  VACCINE    AND    SERUM   THERAPY. 

a  rubber  teat  which  is  attached  to  the  large  end  of  the  pipette, 
leiicocytic  cream  is  drawn  into  the  capillary  end  up  to  the  mark. 
Then  the  capillary  end  is  withdrawn  from  the  leucocytic  emulsion 
and  the  emulsion  in  the  tube  is  drawn  about  one  quarter  inch 
further  up  into  the  tube.  After  this  the  capillary  end  of  the 
pipette  is  immersed  in  the  bacterial  emulsion,  which  is  taken  into 
the  tube  up  to  the  mark.  The  tube  is  withdrawn  and  again  a 
small  amount  of  air  drawn  into  the  capillary  tube.  After  this  the 
serum  is  drawn  in,  again  taking  the  amount  necessary  to  fill  the 
capillary  pipette  to  the  mark.  Walker  has  suggested  that  small 
quantities  of  serum  and  leucocytic  cream  be  put  into  small  tubes 
and  that  serum  and  leucocytes  be  taken  only  once  from  each  of 
these  small  tubes.  This  is  done  in  order  to  avoid  carrying  ma- 
terials from  one  ttibe  into  another. 

The  capillary  pipette  now  contains  equal  volumes  of  leuco- 
cytic cream,  bacterial  emulsion,  and  serum,  as  is  show^n  in   Fig.  9. 


Fig.  S. 


Fig.  9. 

All  three  volumes  are  now  forced  out  onto  a  glass  slide,  being  drawn 
in  and  out  with  the  pipette  in  order  to  mix  the  three  parts  thor- 
oughly. When  this  has  been  done  the  mixture  is  drawn  into  the 
tube  and  allowed  to  come  about  half  way  up  the  capillary  part 
when  the  open  end  is  sealed  off  in  a  pilot  flame  of  the  Bunsen 
burner.  Walker  mixes  the  three  factors  in  a  small  test  tube 
taking  precautions  against  air  bubbles.  Slides,  however,  have 
seemed  better  adapted  especially  because  air  bubbles  are  more 
easily  avoided. 

After  sealing  the  capillary  pipette,  containing  the  mixture  of 
leucocytes,  bacteria  and  serum,  it  is  incubated  at  37°  C.  Realiz- 
ing that  the  pipette  containing  the  mixture  will  not  assume  the 
temperature  of  the  thermostat  if  it  is  merely  exposed  to  the  air 
in  the  thermostat,  Wright  originated  a  so-called  opsonizer. 

The  length  of  time  for  which  the  mixture  is  incubated  varies 
for  the  different  organisms.     It  is  to  be  noted  that  up  to  a  certain 


wSMEARS.  33 

limit  the  longer  the  period  of  incubation  the  more  marked  will 
be  the  phagocytosis.  The  incubation  time  must  in  some  cases 
be  limited  because  of  solution  and  agglutination  of  bacteria. 
In  all  instances  the  mixture  containing  patient's  serum  and 
that  containing  normal  serum  must  be  incubated  at  the  same 
temperature  and  for  the  same  length  of  time.  The  time  of  in- 
cubation varies  from  five  to  thirty  minutes,  depending  upon  the 
species  of  microorganism  and  the  properties  of  the  blood  serum 
tested. 

After  incubation  the  nipple  is  removed  and  the  capillary 
end  of  the  pipette  is  broken  off,  the  nipple  is  then  replaced  and 
the  leucocytes,  bacteria  and  serum  are  again  thoroughly  mixed 
on  a  glass  slide,  after  which  smears  are  made. 

SMEARS. 

If  reference  again  is  made  to  the  principle  involved  in  the 
determination  of  the  opsonic  index,  it  will  be  noted  that  the  index 
shows  the  ratio  of  the  number  of  bacteria  taken  up  by  the  poly- 
morphonuclear neutrophiles  w^hen  patient's  serum  is  a  part  of 
the  mixture,  to  the  number  of  bacteria  taken  up  by  the  same 
class  of  leucocytes  when  normal  serum  is  a  part  of  the  mixture. 
It  is  therefore  necessary  to  count  the  number  of  bacteria  in  the 
polynuclear  leucocytes.  In  order  to  have  an  abundance  of  leuco- 
cytes the  grayish  red  layer  is  taken  off  the  centrifuged  blood. 
This  contains  a  relatively  larger  number  of  white  blood  cells  than 
does  blood  freshly  drawn.  If  a  drop  of  this  leucocytic  cream 
is  spread  out  by  dropping  a  cover  glass  onto  it,  fixed,  and  stained, 
many  red  blood  cells  and  some  w^hite  blood  cells  wall  be  found  in 
the  field  of  the  microscope.  It  w^ould,  how^ever,  be  a  tedious 
task  to  find  fifty  or  one  hundred  polynuclear  neutrophiles.  Wright 
and  Douglas  have  devised  an  ingenious  method  to  gather  the  leuco- 
cytes together  in  certain  parts  of  the  slide.  The  method  of  doing 
this  is  based  on  the  difference  in  size  of  red  blood  cells,  mononu- 
clear and  polynuclear  leucocytes;  the  polynuclear  and  large  mon- 
onuclear cells  being  largest  in  size. 

The  method  of  Wright  and  Douglas  is  as  follows:  A  slide  is 
cleaned  and  slightly  scratched  by  rubbing  with  jeweler's  emory 
paper  to  slightly  roughen  the  surface  of  the  glass  slide.  On  the 
left  end  of  this  slide  a  drop  of  the  incubated  mixture,  which  has 


34 


VACCINE    AND    SERUM   THERAPY. 


been  thoroughly  mixed  after  incubation,  is  placed.  Then  a 
slide  with  a  smooth  edge  is  made  into  a  spreader  by  breaking 
off  a  corner.  This  is  done  so  that  the  two  margins  of  the  spread 
shall  be  on  the  slide  instead  of  running  over  the  edges  of  the  same. 


Fig.  10. 

The  narrowed  end  of  the  spreader  is  now  touched  to  the  slide 
and  the  drop  of  blood  allowed  to  run  to  the  under  edges  of  the 
spreader.  The  spreader,  held  at  an  angle  of  about  35°  to  45° 
is  drawn  over  the  slide  as  is  indicated  in  Fig.  10.  The  spread 
on  the  slide  will  have  an  outline  as  is  indicated  in  Fig.  11. 

The  different  steps  taken  in  making  the  spread  are  essential 
to  secure  good  smears.  When  the  spreader  is  pressed  firmly 
enough  against  the  slide  while  it  is  being  drawn  over  the  same, 


Fig.  11. 


the  polynuclear  leucocytes,  being  larger  than  the  red  blood  cells, 
will  slide  out  at  the  edge  of  the  spreader  or  be  drawn  to  the  end 
of  the  spread.  Care  must  be  taken  not  to  make  the  smear  too 
thin. 


FIXING  AND  STAINING  OF  THE  SMEARS. 

After  the  smear  has  been  made  it  must  be  fixed  and  stained 
for  examination.  Fixing  and  staining  is  usually  accomplished 
by  the  ordinary  blood  stains,  Leishman's,  Wright's,  Jenner's  all 
giving  good  results, except  when  the  index  for  the  tubercle  bacillus 
is  to  be  determined.     The  writer,  however,  has  gotten  more  sat- 


CALCULATION    OF    OPSONIC    INDEX.  OO 

isfactory  results  for  organisms,  other  than  the  bacillus  of  tuber- 
culosis, by  fixing  the  spread  with  methyl  alcohol  for  one  minute, 
washing  off  with  water  and  then  staining  with  Loeffler's  methy- 
lene blue  for  three  to  five  minutes.  In  staining  slides  on  which 
the  index  for  the  tubercle  bacillus  is  to  be  obtained,  a  modification 
of  Wright's  method  has  given  most  satisfactory  results.  The  smears 
are  first  fixed  in  a  saturated  solution  of  bichloride  of  mercury  which 
is  then  washed  oft'  with  water.  After  this  the  slide  is  immersed 
in  a  jar  containing  Ziehl's  carbol-fuchsin .  The  stain  is  heated 
by  placing  the  jar  in  a  heated  water  bath.  After  five  minutes 
of  staining  in.  hot  carbol-ftichsin,  the  stain  is  washed  off  and  the 
smear  is  decolorized  in  a  mixture  of  97  parts  of  alcohol  and  3 
parts  of  concentrated  hydrochloric  acid.  The  smear  is  counter 
stained  in  a  solution  of  one-half  grams  each  of  sodium  carbon- 
ate and  methylene  blue  in  100  c.  c.  of  water.  This  counter-stain 
acts  rapidly  and  has  the  advantage  that  if  the  slide  be  over 
stained  in  the  process  it  can  be  easily  decolorized  with  warm  water. 

EXAMINATION  OF  SMEARS. 

The  spreads  are  examined  by  first  going  over  the  slide  with 
the  low  power  of  the  microscope  to  determine  whether  the  slide 
has  been  properly  stamed  and  also  to  find  the  part  of  the  slide 
where  the  polynuclear  leucocytes  are  most  numerous.  Usually 
it  is  found  that  these  are  most  abundant  near  the  margins  and 
at  the  end  of  the  smear.  Wright  has  recommended  that  those 
at  the  end  of  the  smear  be  examined. 

Under  the  high  power  of  the  microscope  the  number  of  bac- 
teria engulfed  by  a  definite  number  of  polynuclear  leucocytes  is 
determined.  The  number  of  leucocytes  examined  varies.  Wright 
based  many  of  his  determinations  on  examinations  of  twenty 
leucocytes.  Most  investigators,  however,  have  counted  the  num- 
ber of  bacteria  in  fifty  or  more  polynuclear  leucocytes. 

The  average  number  of  bacteria  per  leucocytes  is  spoken 
of  as  the  phagocytic  index. 

CALCULATION  OF  THE  OPSONIC  INDEX. 

The  opsonic  index  of  Wright  is  determined  by  dividing  the 
phagocytic    index    obtained  when  patient's  serum  is  used  with 


36  VACCINE    AND    SERUM   THERAPY. 

a  certain  leucocytic  cream  and  bacterial  suspension,  by  the  pha- 
gocytic index  obtained  when  serum  from  the  healthy  individual 
is  used  with  the  same  leucocytic  cream  and  bacterial  emulsion. 
This  may  be  illustrated  in  a  concrete  case  as  follows :  If  the  aver- 
age number  of  staphylococci  phagocytosed  as  determined  by 
counting  the  cocci  in  fifty  leucocytes  is  eight  when  the  patient 
serum  is  used,  and  ten  when  normal  serum  is  used,  then  eight- 
tenths  or  0 . 8  is  the  opsonic  index. 


CHAPTER  IV. 


CRITICISMS  AND  MODIFICATIONS 

OF  WRIGHT'S  OPSONIC  INDEX 

DETERMINATIONS 


Wright's  publications  on  opsonins,  the  part  they  play  in  im- 
munity and  the  method  of  determination  of  the  opsonic  index 
lead  to  much  investigation  on  these  subjects.  Many  mvestiga- 
tors  followed  Wright's  methods  closely  while  some  have  followed 
what  they  supposed  were  Wright's  methods.  The  first  reports 
of  work  done  by  investigators  other  than  Wright  and  his  pupils, 
agree  strikingly  with  the  results  obtained  by  Wright.  Later 
results,  however,  were  not  as  favorable  either  before  or  after 
Wright  himself  and  some  of  his  pupils  had  demonstrated  and 
taught  his  technique.  For  a  time  want  of  personal  skill  and 
ability,  improper  methods  of  work,  and  lack  of  ability  to  manipu- 
late were  supposed  to  account  for  results  which  did  not  agree 
with  those  obtained  by  Wright.  Numerous  investigators,  how- 
ever, after  having  received  instructions  from  Wright  and  his 
pupils  on  the  methods  of  determining  the  opsonic  index,  seriously 
questioned  the  methods  of  Wright.  This  list  of  investigators 
includes  Park,  Simon,  Baldwin,  Cole,  Moss,  Potter,  Bolduan, 
Walker,  and  many  others.  On  the  other  hand  there  are  num- 
erous investigators  who  have  not  questioned  the  technique  and  the 
results  obtained  by  following  the  methods  of  Wright.  These 
investigators  followed  principally  the  opsonic  index  in  the  various 
bacterial  diseases  and  made  efforts  to  determine  the  nature  of 
opsonins,  their  importance  and  behavior  in  the  various  diseases. 

The  opsonic  index,  according  to  Wright,  is  obtained  when 
the  average  number  of  bacteria  ingested  per  leucocyte  in  a  mix- 
ture of  patient's  serum,  bacterial  emuslion  and  leucocytes  in- 
cubated for  a  certain  period  of  time,  is  divided  by  the  average 


38  VACCINE    AND    SERUM    THERAPY. 

number  of  bacteria  ingested  !)y  the  leucocytes  in  a  mixture  of 
the  same  quantity  of  serum  from  a  normal  individual,  the  same 
bacterial  emulsion  and  leucocytic  cream,  incubated  for  the  same 
length  of  time  at  the  same  temperature.  In  the  process  of  the 
determination  of  the  opsonic  index  mechanical  technique,  serum, 
bacterial  emulsion,  leucocytic  cream,  and  calculation  of  the  aver- 
age number  of  bacteria  ingested  are  of  importance.  Inasmuch 
as  the  results  obtained  by  the  different  investigators  are  so 
discordant  and  inconsistent,  all  of  these  factors  have  been 
considered,  emphasized  and,  in  the  minds  of  some  at  least, 
improved. 

MECHANICAL   TECHNIQUE. 

The  mechanical  part  of  the  technique  of  Wright  has  prob- 
ably been  less  criticised  than  any  other  part  of  his  method.  Var- 
ious investigators  have  reported  their  technique  in  full  and  some 
at  least  have  shown  that  they  have  not  interpreted  Wright's  ex- 
planations of  the  same  correctly,  while  others  have  modified  the 
technique  in  order  to  give  greater  accuracy  to  the  method  of  the 
determination  of  the  index. 

Only  some  of  the  modifications  suggested  can  be  mentioned. 
Barber,  thinking  that  the  mark  on  the  pipette  as  made  by  the 
wax  pencil,  is  too  wide  and  therefore  leads  to  inaccuracies  in  the 
amounts  used  in  the  mixtures,  has  suggested  that  the  mark  on 
the  capillary  pipette  be  made  with  a  glass  hair  dipped  in  Bismarck 
black.  This  suggestion,  however,  is  not  generally  followed  as  it 
is  possible  for  one  experienced  in  handling  the  capillary  pipette 
to  draw  fluids  to  the  upper  or  lower  edge  of  the  wax  pencil  mark 
with  as  much  accuracy  as  it  is  to  take  it  to  the  mark  made  by  the 
glass  hair. 

Walker  has  suggested  the  use  of  a  water  bath  for  the  incu- 
bation of  the  mixture  of  serum,  leucocytes,  and  bacterial  emul- 
sion. Undoubtedly  this  has  advantages  over  incubation  for 
short  periods  of  time  in  the  air  in  the  thermostat.  Wright's  op- 
sonizer,  however,  is  more  convenient  and  almost  immediately 
brings  the  pipette  to  the  temperature  of  the  metal  of  the  opsonizer. 

For  the  collection  of  serum.  Walker  has  suggested  that  the 
capsule  be  held  in  position  by  inserting  it  into  a  slit  in  a  slide 
box.      Unless    one     is    taking    his    own    blood,    however,    it     is 


NORMAL    SERUM.  39 

about  as  convenient  to  hold  the  capsule  in  the  hand  as  to  hold 
the  punctured  finger  near  the  table  on  which  the  slide  box  rests. 

Walker  has  also  advocated  that  small  quantities  of  full 
strength  or  diluted  sera  and  leucocytic  cream  be  put  into  separ- 
ate small  test  tubes  in  order  to  avoid  carrying  bacterial  emulsion 
to  the  lencocytic  cream  from  which  other  preparations  are  to  be 
made  later.     This  method  is  certainly  to  be  recommended. 

Walker  has  also  modified  Wright's  methods  concerning  the 
capillar}^  pipette  containing  the  mixture.  In  making  opsonic 
index  determinations,  Walker  breaks  off  the  large  end  of  the  cap- 
illary pipette  and  seals  both  ends  of  the  capillar}^  tube.  When 
this  is  done  it  becomes  necessary  to  again  fit  the  capillary  part 
of  the  pipette  to  a  larger  tube  to  which  a  rubber  teat  can  be  fas- 
tened. This  Walker  accomplishes  by  fastening  a  thin  rubber 
sheet  over  the  open  end  of  the  large  pipette.  Thisworks  as  a  dia- 
phragm and  can  be  easily  punctured  by  the  capillary  tube. 

SERUM. 

Immunity  to  and  recovery  from  numerous  infections  accord- 
ing to  Wright's  theory  is  due  to  the  presence  of  opsonin.  Opsonin 
prepares  bacteria  for  ingestion  by  the  leucoc5rtes  and  is  a  sub- 
stance contained  in  the  serum.  In  the  determination  of  the 
opsonic  index  the  ratio  of  bacteria  ingested  by  the  leucocytes  is 
established  by  mixing  bacteria  and  leucocytes  in  one  case  with 
patient's  serum  and  in  another  case  with  serum  from  healthy 
individuals.  It  is  thus  evident  that  the  serum  is  the  important 
and  valuable  factor. 

Normal  Serum. 

In  the  determination  of  the  opsonic  index  numerous  in- 
vestigators have  been  struck  by  the  apparently  unexplainable 
indices  found.  This  has  lead  to  the  investigation  of  the 
opsonic  index  obtained  with  serum  from  the  normal  individual, 
to  deterinine  whether  the  index  does  not  vary  in  the  normal,  as 
well  as  in  the  infected,  individual. 

If  reference  is  made  to  the  technique,  as  proposed  to  obtain 
the  normal  phagocytic  index,  it  will  be  observed  that  Wright 
recognized  that  there  is  a  difference  in  the  amount  of  opsonin 
present   in  serum  from  the  normal  individual.     He  states  that 


40  VACCIXE    AND    SERUM   THERAPY. 

the  opsonic  index  of  a  normal  individual  will  vary  from  O.S  to 
1.2.  To  obtain  the  true  amount  of  opsonin  in  normal  serum,  he 
pools  equal  quantities  of  sera  from  several  normal  individuals. 
However,  after  it  has  been  established  that  a  healthy  individual 
has  an  opsonic  index  of  1.0,  as  determined  by  estimation  with 
pooled  normal  sera,  he  uses  serum  from  this  individual  as  a  normal 
serum.  Wright's  own  determinations,  from  which  he  concedes 
that  there  are  variations  in  the  amount  of  opsonin  present  in 
the  blood  of  healthy  individuals,  discredit  the  accuracy  either 
of  the  method  of  determining  the  opsonic  index  or  the  A^alue  of 
determining  the  same.  If  the  opsonic  indices  of  two  normal  in- 
dividuals is  0 . 8  and  1 . 2  respectively  on  the  same  day  as  deter- 
mined by  CO  parison  with  pooled  normal  serum,  then  as  com- 
pared to  each  other  their  opsonic  indices  vaiy  from  0.66  to  1.5 
respectively.  It  is  thus  evident  that  from  Wright's  concession 
the  index  of  a  normal  individual  may  vary  from  0 .  66  to  1.5 
when  one  serum  is  used  as  a  control  for  the  other. 

Indices  for  normal  serum  as  actually  determined  will  not 
even  vary  within  these  limits.  The  writer  sometime  ago  reported 
results  on  a  series  of  ten  specimens  of  blood  from  ten  healthy 
adults,  studied  to  determine  the  differences  in  opsonic  power  in 
healthy  individuals.  The  leucoc3rtes  were  washed  twice  with 
0.85  salt  solution.  The  number  of  staphylococci  taken  up  by 
100  polynuclear  leucocytes  was  counted  in  two  preparations,  sepa- 
rately incubated.  The  phagocytic  indices  as  determined  by  two 
workers  A  and  B  are  as  follows: 

Source.  A  B 

N 9.48  7.63 

J 9.16  5.20 

B 8.75  6.80 

McL 7 .  27  4.37 

Z 6.63  5.12 

C 6.60  5.37 

S 5.89  4.82 

0 5.84  4.57 

H 5.49  6.03 

Ns 4.91  4.37 

The  great  error  introduced  by  this  method  becomes  evident 
upon  the  determination  of  the  opsonic  indices  when  each  serum 
is  used  for  comparison  with  all  of  the  others.  In  the  series  A, 
the  normal  opsonic  index  varies  from  0.  52  to  1 .  93,  while  in  series 
B  it  varies  from  0.57  to  1.75.  Moreover,  the  indices  obtained 
by  the  two  workers  A  and  B  vary  considerably  for  the  same  sera. 


DILUTIONS    OF    SERUM.  41 

Bolduan  has  reported  determinations  of  the  opsonic  index 
of  normal  individuals  and  finds  that  as  far  as  the  bacillus  of  tuber- 
culosis, staphylococci  and  streptococci  are  concerned,  there  is 
marked  variation  not  only  in  different  individuals  but  also  from 
day  to  day. 

Moss  has  found  opsonic  indices  for  staphylococci  to  vary 
from  0.18  to  0.56  in  normal  rabbits. 

The  same  serum  has  also  been  tested  a  number  of  times 
with  the  same  leucocytes  and  bacterial  emulsion.  Bolduan  re- 
ports opsonic  index  determinations  for  a  single  serum  which 
vary  from  1 .  05  to  1 .  46.  The  writer  has  reported  determinations 
made  by  taking  five  specimens  of  blood  from  the  five  fingers 
of  the  same  hand  of  a  healthy  adult.  The  leucocytes  used  for 
the  determination  were  washed  twice  in  1.5  per  cent  potassium 
citrate  in  normal  salt  solution.  The  phagocytic  indices  for 
Mic.  pyogenes  aureus  are  shown  in  the  table: 

1st  100  leukocytes     2d  100  leukocj'tes     Phagocytic 

counted.  counted.  index. 

Specimen    1 166  196  1.810 

2 176  183  1.795 

3 198  199  1.985 

4 185  175  1.800 

5 230  190  2.100 

If  the  indices  for  each  specimen  of  blood  be  compared  with 
all  the  others,  the  opsonic  index  as  determined  by  counting  the 
number  of  cocci  in  two  hundred  cells  will  be  found  to  vary  from 
0.85  to  1.22. 

It  thus  seems  evident  that  if  normal  serum  varies  as  much 
as  has  been  found,  the  variations  from  the  normal,  unless  marked, 
can  indicate  but  little.  The  most  that  would  seem  warranted 
is  to  determine  whether  a  serum  is  "high"  or  "low"  in  opsonic 
content,  as  the  case  may  be. 

Dilutions  of  Serum. 

Wright  has  usually  used  undiluted  serum  in  the  determina- 
tion of  the  opsonic  index.  Simon,  Lamar  and  Bispham  have 
shown  that  by  the  dilution  of  some  sera  there  often  is  a  rapid 
exhaustion  of  phagocytic  power  of  the  leucocytes.  They  have 
observed  that  undiluted  pig's  serum  manifests  a  most  intense 
opsonizing  effect  on  staphylococci  but  that  this  action  diminishes 
markedly  upon  dilution  of  the  serum.  Human  serum,  on  the 
other  hand,  though  not  having  as  marked  an  initial  opsonizing 


42  VACCINE    AND    SERUM    THERAPY. 

power,  will  retain  this  power  on  much  greater  dilution.  They 
liave  fiirther  found  that  in  human  beings,  who  are  supposedly 
in  good  health,  the  phagocytic  power  in  concentrated  serum  will, 
in  some  cases,  diminish  in  proportion  to  the  degree  of  dilution, 
while  with  the  serum  of  other  individuals,  more  rapid  exhaustion 
takes  place. 

Moss  has  compared  the  opsonic  index  in  various  dilutions 
of  serum  and  has  found  that  the  index  obtained  in  one  dilution 
is  not  proportional  to  that  obtained  in  all  the  other  dilutions. 

Walker's  reports  on  the  results  obtained  by  the  dilution  of 
serum  differ  from  those  obtained  by  Moss.  Walker  has  found 
that  wHth  many  bacteria,  undiluted  normal  serum  will  opsonize 
so  many  bacteria  in  a  heavy  suspension  of  bacteria  that  the  leu- 
cocytes will  contain  so  many  microorganisms,  that  counting  is 
impossible.  If  the  bacterial  suspension  be  made  less  heavy  fewer 
bacteria  will  be  taken  up  by  the  leucocytes,  but  under  this 
condition  the  serum  will  not  be  exhausted  of  its  opsonin  and 
part  will  be  lost  in  the  estimation  of  the  opsonic  index.  A 
serum  containing  much  less  opsonin  may  opsonize  just  as  many 
bacteria  in  a  light  bacterial  suspension  as  a  serum  containing 
more  opsonin.  "When  thin  suspensions  of  bacteria  for  which 
much  opsonin  exists  in  the  serum  are  used  it  generally  happens 
that  both  the  sera  sensitize  all  the  bacteria  so  that  the  work  if 
accurately  done  will  produce  ecivial  phagocytic  indices  for  both — 
in  other  terms,  an  opsonic  index  of  unity — regardless  of  the  real 
relation  of  the  serum.  Phagoc^^tic  indices  proportional  to  the 
sera  tested  may  readily  be  obtained  by  diluting  all  the  sera  equally 
to  a  sufficient  degree,  and  using  with  these  diluted  sera  a  thick 
bacterial  suspension."  For  some  bacteria  as  B.  typhosus,  cer- 
tain strains  of  streptococci  and  tubercle  bacilli,  Walker  does  not 
recommend  the  dilution  of  serum,  while  for  staphylococci, 
some  strains  of  streptococci  and  tubercle  bacilli  and  for  B.  coli 
he  does. 

Walker's  views  concerning  the  utilization  of  all  the  opsonin 
capable  of  sensitizing  the  particular  species  of  bacteria  by  the 
use  of  heavy  bacterial  suspension  and  diluted  sera,  must  be  favor- 
ably received.  Before  they  can  be  accepted  and  followed  it  will, 
however,  be  necessary  to  disprove  the  results  of  Moss  and  others 
concerning  the  dilution  of  opsonin  by  the  dilution  of  the  serum. 


BACTERIAL    EMULSIONS.  43 

BACTERIAL   EMULSIONS. 

Wright,  in  his  technique,  attempts  in  every  case  to  break 
up  all  clumps  of  bacteria  and  tries  to  get  such  strength  of  suspen- 
sion that  the  number  of  bacteria  per  leucocyte  can  be  easily 
determined.  With  certain  organisms  trial  tests  are  necessary. 
After  this,  the  suspension  may  be  diluted  or  bacteria  added  as 
may  be  indicated. 

Most  investigators  have  experienced  difficulty  in  obtaining 
suitable  suspensions  and  Wright  himself  has  changed  his  tech- 
nique for  making  bacterial  suspensions  from  time  to  time.  The 
difficulties  that  have  been  experienced  are  of  two  kinds:  those 
dealing  with  the  strength  of  the  bacterial  suspension,  and  those 
concerning  the  condition  of  the  microorganisms  in  the  suspension. 

Difficulties  depending  upon  the  strength  of  the  bacterial 
suspension.  Numerous  investigators  have  given  definite  figures 
to  be  obtained  for  the  phagocytic  index  with  normal  serum.  In 
most  cases  it  is  desirable  that  the  phagocytic  index  for  normal 
serum  be  between  six  and  fifteen.  Simon  has  found  that  when 
the  emulsion  contains  between  666,000  and  2,000,000  microor- 
ganisms per  cubic  millemeter  the  best  results  are  obtained.  Re- 
cently Walker  has  emphasized  certain  facts  that  must  be  taken 
into  consideration  in  determining  the  strength  of  the  bacterial 
emulsion.  If  the  suspension  does  not  contain  enough  bacteria 
to  exhaust  all  the  opsonin  in  the  strongest  serum,  a  certain  amount 
of  opsonin  will,  according  to  Walker,  be  lost  in  the  estimation  of 
the  opsonic  index.  In  order  to  get  the  right  strength  of  bacterial 
suspension.  Walker  tests  the  same  with  serum  diluted  1  to  30 
and  1  to  15.  If  the  1  to  15  dilution  of  serum  shows  a  phagocytic 
index  twice  as  great  as  that  obtained  in  the  1  to  30  dilution  then 
enough  bacteria  are  present  in  the  suspension.  Too  such  a  sus- 
pension he,  however,  adds  more  of  the  culture,  because  having 
more  bacteria  than  are  absolutely  necessary  will  not  change  the 
opsonic  index  but  will  certainly  furnish  enough  bacteria  to  ex- 
haust the  serum  of  its  opsonin.  Only  with  the  staphylococci 
will  a  too  heavy  suspension  effect  the  true  index.  This  Walker 
believes  is  due  to  the  action  of  a  product  of  bacterial  growth. 
To  overcome  the  excessive  phagocytosis  when  heavy  suspensions 
of  bacteria  are  used.  Walker  dilutes  serum.    However,  as  has  been 


44  VACCINE    AND    SERUM   THERAPY. 

mentioned  before,  there  are'conflicting  views  concerning  the  effect 
of  dilution  of  serum  for  which  the  opsonic  index  is  to  be 
determined. 

DIFFICULTIES  ARISING  BECAUSE  OF  THE  PARTICULAR 
SPECIES   OF  BACTERIA  IN  THE   SUSPENSION. 

Anyone  who  has  made  opsonic  index  determinations  has  at 
times  had  sHdes  to  examine  in  which  the  bacteria  were  not 
well  stained  and  in  which  the  microorganisms  were  apparently 
clumped.  Frequently  only  shells  of  organisms  remain  and  at 
times  the  bacteria  are  completely  dissolved.  These  difficulties 
are  apparently  dependent  upon  the  particular  culture  used  but 
to  a  greater  extent  upon  the  agglutinins  and  lysins  present 
in  different  sera.  To  overcome  this  difficulty  young  cultures 
are  preferred  because  they  stain  better  than  do  old  cultures. 
To  overcome  agglutination  and  lysis  the  serum  is  in  some  cases 
heated  to  55°  or  65°.  The  clumping  of  bacteria  in  opsonic 
index  preparations  is  given  little  attention  by  Wright.  Other 
investigators  have,  how^ever,  regarded  clumping  of  bacteria 
as  responsible  for  the  occasional  enormous  differences  of  the 
indices  determined.  Walker  gives  tables  which  indicate  the 
objection  of  clumps  on  the  uniformity  of  phagocytosis  and  has 
prepared  a  technique  by  which  he  obtains  clump-free  bacterial 
suspensions.  According  to  this  technique  the  bacteria  are  rub- 
bed gently  in  a  mortar  with  small,  and  gradually  increasing, 
quantities  of  salt  solution  until  a  thick  suspension  is  obtained. 
This  suspension  is  then  filtered  through  moistened  scraped  filter 
paper  until  in  a  stained  specimen  no  more  clumps  are  found. 
Even  though  a  technique  may  be  devised  to  obtain  relatively 
clump-free  suspensions  of  bacteria,  this  will  not  do  away  with  the 
clumping  occasionally  observed  in  opsonic  index  preparations, 
because  the  action  of  agglutinins  in  undiluted  or  only  slightly 
diluted  sera  w^ill  again  cause  clumping  of  the  bacteria. 

LEUCOCYTES. 

There  has  been  but  little  criticism  of  the  method  of  collection 
of  leucocytes,  as  proposed  by  Wright.  Simon  has  used  0.1  per 
cent  ammonium  oxalate  instead  of  1.0  percent  sodium  citrate 
in  salt  solution.     Some  investigators  have  drawn  the  corpuscles 


CRITICISM   OF   CALCULATION   OF   OPSONIC   INDEX.  45 

off  after  the  first  centrifugalization  while  others  have  attempted 
to  remove  the  serum  of  the  blood  and  the  citrate  or  oxalate 
solution  by  several  washings  with  normal  salt  solution. 

To  the  source  of  the  leucocytes  Wright  attaches  little  impor- 
tance, while  most  other  investigators  have  aimed  to  obtain  leuco- 
cytes froiTL  the  blood  of  apparently  healthy  individuals.  The 
work  of  Peterson  and  Hiss  and  Zinsser  indicates  that  the  leuco- 
cytes contain  substances  that  are  of  importance  in  immunity 
and  that  these  substances  vary  in  the  leucocytes  of  normal  and 
immunized  animals.  For  this  and  other  reasons  it  seems  advis- 
able in  all  cases  to  obtain  the  leucocytes  from  the  blood  of  normal 
individuals. 

Walker  draws  the  blood  from  which  the  leucocytes  are  to 
be  obtained  into  citrate  solution  heated  to  37°  C.  The  leuco- 
cytic  cream  is  kept  at  this  temperature  up  to  the  time  of  mix- 
ture with  serum  and  bacterial  emulsion.  He  believes  this  to  be 
of  great  importance.  In  experiments  performed  by  the  writer 
and  others,  this  has  not  been  found  to  be  of  especial  value  in  the 
production  of  uniform  phagocytosis,  nor  do  Walker's  tables 
show  a  marked  uniformity,  for  the  number  of  bacteria  per 
leucocyte  vary  froin  1  to  1 7  in  twenty-five  leucocytes  examined 
on  the  same  slide. 

CALCULATION  AND  DETERMINATION  OF  THE 
OPSONIC  INDEX. 

According  to  Wright's  method  the  opsonic  index  is  deter- 
mined and  calculated  by  dividing  the  degree  of  phagocytosis  ob- 
tained with  patient's  serum  by  that  obtained  with  serum  from  a 
normal  individual. 

Simon  has  proposed  an  entirely  different  method  for  the 
calculation  of  the  opsonic  index.  He  had  observed  that  with 
many  species  of  bacteria,  due  to  lysis  and  fragmentation,  great 
difficulty  is  experienced  in  determining  the  phagoc3rtic  index  and 
that  the  strength  of  emulsion  greatly  changes  the  amount  of 
phagocytosis.  To  remedy  this,  Simon  first  proposed  that  the 
percentage  of  phagocytic  cells  be  determined  by  counting  50  cells 
taken  in  sequence.  Later  Simon  compared  the  percentage  of 
phagocyting  leucocytes  with  patient's  serum  to  the  percentage 
of  phagocyting  cells  with  pooled  normal  serum.     The  value  ob- 


46  VACCINE    AND    SERUM    THERAPY. 

tained  for  pooled  normal  serum  he  called  1.0,  and  the  index  de- 
termined in  this  way  he  called  the  percentage  index  as  contrasted 
to  Wright's  bacillary  index.  These  two  indices  Simon  finds  can 
be  made  to  agree  to  the  second  decimal  place. 

Moss  and  others  have  found  that  the  absolute  numbers  of 
cells  phagocytosing  depends  in  normal  or  slightly  diluted  sera  on 
the  strength  of  the  bacterial  suspension,  and  that  in  low  dilutions 
practically  all  the  cells  can  be  made  to  engulf  bacteria.  It  is 
evident  that  by  Simon's  method  only  sera  having  low  indices  can 
be  investigated,  unless  the  bacterial  emulsion  or  serum  are  di- 
luted. Simon  has  recommended  that  light  bacterial  suspensions 
and  at  times  diluted  sera  be  used  so  as  to  be  able  to  determine 
the  percentage  index. 

Strong,  in  his  early  experiments  with  anti-plague  serum, 
determined  the  index  according  to  Wright's  method.  Later, 
however,  he  substituted  this  method  by  one  in  which  the  highest 
dilution  of  an  immune  serum  which  gives  a  marked  phagocytosis 
is  compared  to  the  same  dilution  of  a  normal  serum.  If,  with  a 
certain  dilution  of  an  immune  serum  marked  phagocytosis  is  ob- 
served while  the  same  dilution  of  a  normal  serum  gives  only  slight 
phagocytosis,  the  immune  serum  is  regarded  as  having  an  increased 
opsonin  content.  Even  on  this  method  Strong  places  little  re- 
liance. 

Although  Simon  prefers  to  accept  his  index  to  Wright's 
when  there  is  disagreement  between  the  two,  most  investigators 
have  preferred  Wright's  opsonic  index. 

LEUCOCYTES  IN  WHICH  THE  BACTERIA 
ARE  TO  BE  FOUND. 

According  to  Wright's  method  the  bacteria  are  to  be 
counted  in  the  pohmuclear  neutrophiles.  These  cells  when 
Wright's  technique  is  followed  will  be  found  along  the  margins 
and  end  of  the  spread. 

In  all  work  on  the  determination  of  opsonic  indices  it  has  been 
found  that  there  are  great  differences  in  the  number  of  bacteria 
taken  up  by  the  individual  leucocytes.  Moreover,  great  difTer- 
ences  have  been  found  in  the  phagocytic  index  as  determined  by 
different  investigators  examining  the  same  vslide.     Even  the  same 


LEUCOCYTES    EXAMINED.  47 

investigators  have  obtained  markedly  different  phagocytic  indices 
on  the  same  slide.  The  writer  has  reported  work  showing  that 
there  is  not  always  agreement  between  results  obtained  by  count- 
ing the  bacteria  in  100  and  200  polynuclear  leucocytes  on  the 
same  slide.  Bolduan,  Moss,  Cole,  Jeans  and  Sellards,  Simon  and 
others  have  published  results  showing  striking  differences  in  the 
counts  obtained  in  50,  100,  200  and  more  cells.  Bolduan  supposes 
that  counting  the  number  of  bacteria  ingested  by  100  or  150  cells 
will  give  a  fairly  correct  average.  Potter,  Dittman,  and  Bradley 
believe  counts  in  100  cells  suffice  in  most  cases.  Moss,  in  an  effort 
to  determine  how  many  leucocytes  must  be  examined,  has  de- 
cided that  after  counting  the  bacteria  in  three  hundred  cells  there 
is  still  an  error  of  ten  per  cent ;  and  when  50  cells  only  are  counted 
his  experiments  show  a  variation  of  from  .  8  per  cent  to  30  per 
cent.  Most  investigators  determine  the  ninnber  of  bacteria  taken 
up  by  at  least  100  leucocytes. 

Large  numbers  of  leucocytes  must  be  examined  because  of 
the  variability  of  the  number  'of  bacteria  taken  up  by  the  indi- 
vidual leucocytes.  Cells  which  are  apparently  normal  as  far  as 
can  be  determined  by  staining  and  which  may  contain  an  average 
of  from  5  to  8  bacteria  per  leucocyte,  will  phagocyte  from  20  to 
oO  microorganisms  at  a  inaximum  while  other  leucocytes  may 
take  up  none.  This  difference  in  the  number  of  bacteria  taken 
up  by  individual  leucocytes  has  been  supposed  to  be  due  to  the 
leucocytes  themselves  and  to  clumps  in  the  bacterial  emulsion. 
Walker  has  suggested  that  the  leucocytes  be  kept  constantly  at  a 
temperature  of  37°  C,  and  that  they  be  well  mixed  after  incuba- 
tion with  serum  and  bacterial  emulsion.  Undoubtedly  all  in- 
vestigators have  mixed  the  leucocytes,  bacteria  and  serum  well 
after  incubation  and  before  making  the  spreads  to  be  examined. 
This  procedure,  however,  does  not  overcome  the  differences  m 
numbers  of  bacteria  per  leucocyte,  as  is  shown  in  Walker's  tables, 
in  which  the  variations  in  numbers  of  bacteria  ingested  range 
from  1  to  15,  2  to  16,  1  to  17,  and  4  to  16  in  four  sets  of  25  leuco- 
cytes examined.  It  cannot  be  concluded  from  this  that  there  is 
a  uniformity  of  phagocytosis,  although  Walker's  tables  do  show 
a  uniformity  of  phagocytic  index  in  groups  as  small  as  25  leuco- 
C5rtes.  It  is  evident,  however,  that  if  25  leucocytes  only  were 
to  be  examined,  the  opsonologist  might,  after  he  had  examined 


48  VACCINE   AXD    SERUM    THERAPY. 

24  leucocytes,  have  some  choice  for  the  25th  one  to  he  examined, 
which  choice  would  change  the  result  from  1  to  17.  Based  on 
this  selection  the  opsonic  index  could  be  materially  lowered  or 
raised  as  might  be  desired. 

In  one  of  the  early  contributions  on  the  subject  of  opsonic 
index,  Simon,  Lamar,  and  Bispham  recommend  that  in  making 
the  spread  of  the  mixture  after  incubation,  the  spreader  slide  be 
merely  kept  in  contact  with  the  blood  without  touching  the  lower 
slide,  for,  "Otherwise  it  may  happen  that  most  of  the  leucocytes 
containing  organisms,  are  carried  to  one  end,  while  only  the  empty 
cells  are  found  in  the  intervening  space."  In  the  laboratories 
of  the  Johns  Hopkins  Hospital,  Cole,  Moss,  and  Jeans  and  Sellard 
have  made  determinations  of  pliagocytic  indices  determined  in 
different  parts  of  the  slide.  It  was  found  that  the  leucocytes 
collected  near  the  edge  of  the  smear  contain  decidedly  more  bac- 
teria than  those  toward  the  center  of  the  slide.  By  dividing  a 
slide  into  three  zones  they  found  that  at  the  end  of  the  smear  the 
leucocytes  contain  more  bacteria,  than  in  the  first  and  middle 
zones.  To  explain  these  differences  these  men  assume  that  the 
polymorphonuclear  leucocytes  containing  the  largest  number  of 
bacteria  are  so  increased  m  size  that  they  are  drawn  to  the  end 
of  the  slide  while  the  smaller  ones  drop  out  earlier.  These  results 
indicate  that,  contrary  to  Wright's  suggestion,  one  ought  not  to 
determine  the  number  of  bacteria  at  the  end  of  the  spread. 

Wright's  method  of  the  determination  of  the  opsonic  index 
has  yielded  such  marked  differences  in  results  that  more  work  on 
this  subject  seems  justifiable.  It  must  be  remembered  that  the 
opsonic  index  is  of  no  value  whatever  unless  carried  out  with  the 
greatest  care  and  by  some  one  who  has  had  considerable  exper- 
ience. Modifications  of  Wright's  technique  have  arisen  with  too 
much  rapidity  to  make  it  possible  to  lay  down  any  definite  rule 
for  the  determinattion  of  the  opsonic  index.  However  there  are 
only  few  modifications  that  have  made  it  possible  to  get  more 
reliable  determinations  of  Wright's  opsonic  index. 


CHAPTER  V. 


OPSONIC  INDEX   IN   HEALTH  AND 

DISEASE. 


The  active  participation  and  function  of  the  white  blood  cells 
in  the  protection  of  the  body  against  infection  was  first  carefully 
studied  by  Metchnikoff.  He  regarded  the  leucocytes  as  cells 
whose  object  is  to  remove  from  the  body  and  its  tissues  bacteria 
and  foreign  material.  On  this  he  based  his  theory  of  immunity. 
It  is  not  strange  that  Leishman,  Wright,  and  others  should  have 
suspected  differences  in  phagocytosis  in  health  and  disease.  In 
1902  Leishman,  and  especially  Wright,  emphasized  the  difference 
in  piiagocytic  ratio  observed  in  individuals  with  infections  run- 
ning favorable  or  unfavorable  courses.  While  trying  to  find  a 
method  by  which  to  control  the  administration  of  vaccine,  Wright 
discovered  opsonins.  With  the  discovery  of  opsonin  and  the 
development  of  a  method  for  the  determination  of  the  same,  it 
was  evident  that  the  opsonic  index  in  health  and  disease  should 
be  investigated.  Wright  found  that  in  individuals  with  localized 
or  chronic  infections  there  is  usually  a  decrease  in  the  opsonin 
content  of  the  blood.  This  decreased  opsonin  content  as  deter- 
mined by  the  opsonic  index,  according  to  Wright's  theories,  is 
due  to  the  fact  that  in  local  infections  but  little  of  the  bacterial 
substance  is  absorbed,  and  so  gives  rise  to  but  a  small  amount  of 
active  immunity.  When  this  is  the  case  the  local  infection  be- 
comes chronic. 

By  the  method  of  the  determination  of  the  opsonic  index, 
Wright  has  demonstrated  that  following  the  injection  of  vaccines 
made  from  the  cultures  of  infecting  organisms,  there  is  first  a 
drop  in  the  opsonic  index  and  later  a  rise  of  the  same.  The  drop 
in  the  index  he  calls  the  "negative"  phase  and  the  rise  the  "posi- 
tive" phase.  The  observation  of  negative  and  positive  phases 
was    made   by   Ehrlich   in    1892,  who   found   that   following   in- 


50  VACCINE    AND    SERUM    THERAPY. 

jection  of  tetanus  toxin  there  is  at  first  a  slight  diminution  in  the 
anti-toxin  in  the  milk  of  the  animals  injected  while  later  there 
is  an  increase  in  the  amount  of  anti-toxin.  Likewise  Solomonson 
and  Madsen  found  that  similar  results  are  produced  in  the  blood 
when  diphtheria  toxin  is  injected.  Agglutinins,  precipitins,  bac- 
teriolysins,  after  injection  of  killed  cultures  of  bacteria,  are  also 
at  first  decreased  and  later  increased. 

Wright  and  Douglas  did  not,  as  has  been  supposed  by  many, 
find  that  all  pathogenic  bacteria  are  sensible  to  opsonins.  They 
divide  the  pathogenic  bacteria  into  four  groups: 

a.  Those  bacteria  that  are  sensible  to  bactericidal,  bacter- 
iol3rtic  and  opsonic  action  of  the  normal  human  blood.  B.  ty- 
phosus and  Spir.  cholerse  belong  to  this  group. 

b.  Those  bacteria  that  are  sensible  to  opsonic  and  especially 
bactericidal  action  of  normal  blood  serum.  Examples  of  this 
group  are  to  be  found  in  B.  coli  and  B.  dysenteriae. 

c.  Those  bacteria  that  are  definitely  sensible  to  opsonin 
but  not  to  the  bactericidal  action  of  normal  blood.  B.  pest  is, 
Mic.  melitensis  and  Mic.  pneumonias  belong  to  this  group. 

d.  Those  bacteria  that  are  insensible  to  the  opsonic  and  bac- 
tericidal action  of  normal  human  blood  serum.  Examples  of 
this  group  are  found  in  the  bacillus  of  diphtheria. 

Practically  all  of  Wright's  observations  on  opsonic  immuni- 
zation were  determined  in  man.  In  the  human,  following  the 
injection  of  killed  cultures,  the  negative  phase  is  visually  of  short 
duration,  lasting  from  24  to  48  hours.  The  positive  phase  may 
not,  but  usually  follows  the  negative  phase  and  lasts  from  3  to 
4  days  to  two  weeks.  In  active  immunization  Wright  reasons 
the  curve  of  protected  substances  may  be  affected  in  different 
ways.  If  the  dose  of  vaccine  injected  is  very  small,  the  negative 
phase  may  be  omitted,  while  if  a  very  large  dose  is  injected  it  will 
be  marked  and  the  highest  index  in  the  positive  phase  may  not  be 
as  high  as  it  was  at  the  time  of  injection.  Usually  in  active  im- 
munization a  number  of  injections  of  vaccine  must  be  made. 
These  may  produce  one  of  three  effects  on  the  opsonic  index: 

a.  If  injections  are  made  after  the  index  has  returned  to 
the  normal,  the  index  curve  may  show  a  series  of  indices  above 
and  below  the  normal.  This  is  what  happens,  according  to  Wright, 


CURVE  OF  PROTECTIVE  SUBSTANCES. 


51 


when  the  individual   is  allowed  to  recover  completely  between 
injections.     The  curve  is  illustrated  in  Fig.  12A. 

b.  The  injections  may  be  given  at  such  times  that  one  nega- 
tive phase  is  superimposed  upon  another  negative  phase  and  in 
this  way  produce  a  cummulative  effect  in  the  duration  and  degree 
of  the  negative  phase.  This  result  is  obtained  according  to  Wright 
when  active  immunization  is  pushed  rapidly  or  forced.  The  curve 
of  protective  substance  is  shown  in  Fig.  12B. 

c.  The  injection  may  be  made  at  such  times  and  in  such 
amounts  that  there  is  a  summation  of  positive  phases.  This  is 
the  result  sought  for  in  immunization.  The  curve  representing 
the  amount  of  protective  substances  in  this  case  is  represented 
in  Fig.  12C. 


Fig.  12. 

Wright,  in  his  treatment  of  infections  by  the  injection  of  vac- 
cine, tries  to  inject  such  doses  at  such  intervals  of  time  as  to  give 
him  a  continuous  "high  tide  phase."  It  is  to  be  noted,  however, 
that  even  with  the  most  favorable  summation  of  positive  phases 
Wright  has  not  obtained  an  index  as  high  as  4 . 0  except  in  rare  in  - 
stances.  This,  of  course,  is  entirely  out  of  proportion  with  the  amount 
of  other  immune  bodies  produced  by  active  immunization. 

Before  the  principle  and  methods  as  formulated  by  Wright 
can  be  accepted  it  is  necessary  to  decide  (a)  that  opsonins  are  of 


52  VACCINE    AND    SKRUM    THKRAPY. 

importance  in  iminimity  and  that  the  method  of  determjnin!:^  the 
opsonic  index  is  accurate;  (b)  that  in  the  natural  recovery  from 
an  infection  the  opsonic  index  changes,  as  Wright  aims  to  change 
it,  by  the  injection  of  bacterial  vaccine;  and  (c)  that  the  injection 
of  vaccine  produces  the  changes  described  by  Wright. 

IMPORTANCE  OF  OPSONIN  IN  IMMUNITY  AND  ACCURACY 

OF  THE  METHOD  OF  THE  DETERMINATION 

OF  THE  OPSONIC  INDEX. 

Spontaneous  phagocytosis  has  been  described  from  time  to  time. 
With  the  introduction  of  Wright's  opsonin  theory  and  the  develop- 
ment of  the  technique  of  opsonic  index  determination,  phagocytosis 
has  been  found  to  occur,  in  most  cases,  only  when  fresh  serum  is 
present.  Neufeld  and  many  others  have  found  that  contrary  to 
Wright's  statement  phagocytosis  is  not  entirely  dependent  upon 
opsonins,  for  bacteria  with  little  virulence  may  be  taken  up  by 
the  leucocytes  even  though  no  serum  is  present.  The  existence 
of  opsonin,  however,  and  its  relation  to  phagocytosis  can  no  longer 
be  doubted;  the  part  it  plays  in  immunity  has  not  yet  been 
satisfactorily  demonstrated.  Many  of  the  objections  made  to 
Metchnikoff's  phagocytic  theory  of  immunity  present  themselves 
in  the  opsonic  theory  of  immunity.  It  has  been  found  that  highly 
virulent  bacteria  are  not  nearly  as  readily  ingested  by  leucocytes 
as  are  less  virulent  ones.  Bacteria  engulfed  by  leucocytes  are 
not  always  dissolved  and  disposed  of  for  it  has  been  demonstrated 
by  various  investigators  that  certain  species  of  bacteria  produce 
or  secret  substances  that  are  antogonistic  to  and  actually  kill 
leucocytes.  Even  if  it  is  accepted  that  opsonin  plays  an  important 
part  in  immunity,  the  opsonic  index  determined  by  Wright's 
method,  need  not  necessarily  be  an  index  of  the  patient's  im- 
munity to  certain  infectious  microorganisms.  Agglutinins,  bac- 
teriolysins,  and  anti-toxins,  all  of  which  are  substances  better 
known  than  are  opsonins,  are  only  circumstantial  evidence  of 
greater  or  less  importance  in  the  complex  phenomenon  of  immu- 
nity. The  body  has  various  means  of  defense  against  bacterial 
invasion  and  action  so  that  the  assumption  that  all  the  protec- 
tive substances  and  means  against  bacterial  diseases  and  infec- 
tions manifest  themselves  in  sucli  a  manner  that  they  can  1)e  de- 
termined frotn  the  opsonic  index  seems  unreasonable. 


OPSONINS    IN    IMMUNITY.  ol'-, 

In  the  previous  chapter  the  accuracy  of  the  method  for  the 
determination  of  the  opsonic  index  has  been  criticised.  It  is 
certain  that  the  method  is  compHcated,  requires  a  considerable 
amount  of  experience,  and  with  all  the  modifications  and  improve- 
ments in  the  technique  is  still  subject  to  such  great  error  that 
even  in  experienced  hands  the  determinations  of  the  opsonic 
index  must  be  regarded  as  of  doubtful  value. 

CHANGES  IN  THE  OPSONIC  INDEX  IN  NATURAL 
RECOVERY   FROM  AN  INFECTION. 

Wright,  at  various  times,  has  reported  observations  on 
the  phagocytic  reaction  and  opsonic  index  in  infections  running 
favorable  and  unfavorable  courses.  He  has  found  that  recovery 
and  increased  opsonic  index  accompany  each  other.  Hektoen 
has  reported  that  in  pneumonia  the  opsonic  index  is  at  first  low 
and  rises  as  the  patient's  condition  improves  so  that  at  the  crisis 
the  index  is  above  normal.  In  patients  that  have  a  persistently 
low  opsonic  index  in  this  disease  death  usually  follows.  Tunni- 
cliff  has  found  that  in  scarlet  fever  the  opsonic  index  for  strepto- 
cocci is  below  normal  early  in  the  disease  and,  as  the  acute  symp- 
toms subside,  it  rises  above  normal.  Later  on  it  again  becomes 
normal.  Ruediger  has  found  in  erysipelas  a  sharp  rise  in  the 
index  for  streptococci  as  the  temperature  begins  to  fall.  Hamilton 
isolated  pseudo-diphtheria  bacilli  in  75  per  cent  of  cases  of  acute 
otitis  media  and  found  wide  variations  in  the  index  for  this  organ- 
ism in  these  cases.  Clark  has  found  that  the  typhoid  opsonic 
index  drops  before  a  relapse  in  typhoid  fever. 

The  writer  in  a  study  on  the  opsonic  index  in  erysipelas  made 
observations  on  the  changes  in  the  opsonic  index  in  unvaccinated 
patients  to  determine  the  relation  between  the  opsonic  index 
for  Streptococcus  erysipelatos  and  recovery  from,  migration,  re- 
currence and  desquamation  in  erysipelas.  The  most  instructive 
of  these  cases  are  the  two  which  follow: 

Case  I. — S.,  a  man,  aged  thirty-eight  years,  who  had  a  migra- 
tory, recurrent  erysipelas  of  the  face,  ears,  scalp,  and  neck,  was 
admitted  to  the  hospital  on  the  fourth  day  of  his  disease.  When 
his  face  was  involved,  his  index  w^as  0.7;  w^hen  it  was  desquamat- 
ing, the  index  had  risen  to  1.3;  when  his  face  was  again  involved 


54 


VACCINE    AND    SERUM    THERAPY. 


seven  days  later,  the  index  was  1.4;  and  when  it  was  desquamat- 
ing again,  the  index  had  dropped  to  O.S.  When  his  neck  became 
involved,  one  day  after  his  face  began  to  desquamate  for  the 
second  time  his  index  was  1.0;  two  days  later,  with  an  index  of 
1.1,  his  back  became  involved,  and  three  days  later,  with  an 
index  of  3 . 2,  his  shoulders  and  neck  w^ere  again  involved,  and  two 
days  later,  with  index  of  1.1,  tlie  back  and  shoulders  began  to 
desquamate.     (Chart  I.) 


DISEASE  i      ■'     ''      ;   S  y  1«  11  12  13  U  Ij  10  17  18  19  20  21  22  23  21  23  26  27  28  21)  30  31 

Chart  I. — Temperatrue  (unbroken  line)  and  opsonic  index  (broken  line)  in  a  case  of 

erysipelas  (Case  I). 

Case  II. — A.,  a  man,  aged  twenty-four  years,  suffering  w^ith 
erysipelas  of  the  face  and  ears,  was  admitted  on  the  eighth  day  of 
the  disease  with  an  index  of  0.9.  The  next  day  his  temperature 
dropped  and  desquamation  began;  his  index  was  found  to  be  1.1. 
Subsequently  the  index,  though  the  patient  was  perfectly  well, 
remained  below^  unity.     (Chart  II.) 


8-0         10 

TEMPCRATURE 


11        12        13        14, 

UPSONIC   INDEX 


Chart  II. — Temperature  (unbroken  line)  and   opsonic   index  (broken   line)  in  a  case  of 

erysipelas  (Case  II). 

In  these  two  cases  there  has  been  no  constant  change  of  index 
corresponding  with  desquamation  and  recovery. 


OPSONIC  INDEX  IN  RECOVERY  FOR  INFECTION 


55 


To  further  study  the  relation  of  the  opsonic  index  to  the 
course  of  the  disease,  the  indices  of  aU  patients  who  had  received 
no  vaccine  were  tabulated  with  regard  to  the  day  of  the  disease, 
and  an  average  opsonic  index  for  each  day  was  determined. 

Table    Showing   the    Opsonic    Index   at   the    Time   of    Admission,  before   Killed 
Cultures   were   Injected,   and    the   Day   of   the   Disease. 


Day 

of  Disease. 

^ 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

0.6 

1.2 

0.6 

0.7 

0.7 

0.4 

0.9 

0.8 

1.1 

0.6 

1.0 

0.3 

1.1 

1.1 

1.1 

1.0 

1.1 

0.8 

0.9 

0.7 

1.0 

2.4 
1.7 

1.4 
1.5 
0.7 
1.0 
1.2 

0.9 
0.6 
1.1 

0.6 
1.6 

1.3 

1.7 

1.0 

0.45 

1.1 

1.45 

1.1 

0.86 

0.4 

1.05 

0.96 

1.23 

0.6 

0.9 

Average. 


The  composite  chart  indicates  that  erysipelas  causes  an  in- 
crease of  the  opsonic  index  which  reaches  its  maximum  about  the 
third  day  of  the  disease,  and  is  followed  by  a  gradual  fall.  The 
subsequent  course  of  the  chart  represents,  in  very  large  part,  ob- 
servations made  upon  recurrent,  migratory,  and  complicated 
cases.  Ruediger  found  that  the  index  was  high  during  an  attack 
of  erysipelas. 


<!          ^V 

,'                       5.^ 

-       -•                                    ^«           - 

-  .            :  -^--*^  _        -4- 

1  0 .- -is 

JJ         iJri   N 

j  =  =  --  =  ;- 5 --- 

'I                :      i-^i^ 

y                                           ^              ,' 

^          -                             s 

_,'^                                 -     -     A     ,«^'- 

._         _                                        _            -*     ^^_- 

_  i 

X_.X -- 

9       10  '    n 


Chart  III. — To  show  the   average  opsonic  index  and   the  daj^  of  disease  before  killed 
cultures  of  streptococci  were  injected. 

It  is  to  be  noted  that  the  determinations  made  and  repre- 
sented in  the  lastcurve  do  not  represent  the  index  in  any  one  case. 


56  VACCINE    AND    SERUM   THERAPY. 

The  indices  in  individual  cases  are  so  variable  and  show  such  great 
irregularity  that  determinations  of  the  opsonic  index  in  any  case 
give  little  indication  of  the  severity  of  the  disease  and  is  of  no 
value  in  prognosis. 

Most  investigators  have  found  that  in  normal  individuals 
the  curve  of  opsonic  index  is  very  irregular.  Moss  in  thirteen 
observations  on  one  individual  determined  indices  which  varied 
from  0.52  to  1.95  and  in  another  from  0.42  to  1.66.  Bolduan 
reports  observations  in  w^hich  one  patient's  serum  varied  in  opsonin 
content  as  determined  by  Wright's  opsonic  index  from  0.82  to 
2 .  50  in  seventeen  days.  Inasmuch  as  the  opsonic  index  during 
infections  shows  no  greater  variation  than  during  health  it  can 
hardly  be  accepted  that  determinations  of  opsonic  indices  are  of 
much  assistance  in  diagnosis  and  prognosis  in  disease. 

Up  to  the  present,  most  investigators  have  found  that  in  the 
natural  course  of  infection  there  is  no  regular  curve  of  the  opsonic 
index  and  that  in  the  natural  recovery  from  an  infection  the 
changes  in  opsonic  index  are  not  those  which  Wright  attempts 
to  obtain  by  injection  of  killed  cultures  of  bacteria. 

CHANGES    IN   OPSONIC   INDEX    PRODUCED    BY   INJEC- 
TIONS OF  KILLED  CULTURES. 

Wright  has  observed  that  the  injection  of  killed  cultures  is 
usually  followed  by  a  change  in  opsonic  index  for  the  organ- 
isms injected.  If  the  amount  of  killed  cultures  injected  is  large, 
he  states  there  is  a  marked  negative  phase,  after  which  there 
may  be  either  a  marked  or  slight  increase  in  the  opsonic  index. 
If  the  amount  of  killed  cultures  injected  is  small,  the  negative 
phase  may  be  small  or  absent  entirely  and  following  this  there 
may  or  may  not  be  a  positive  phase.  Wright  has  attempted  to 
regulate  injection  of  killed  cultures  so  as  to  get  as  small  a  nega- 
tive phase  and  as  large  a  positive  phase  as  is  possible.  To  accom- 
plish this,  he  has  determined  the  number  of  killed  cultures  in  emul- 
sions and  has  injected  certain  numbers  of  bacteria  at  a  dose,  the 
number  varying  with  the  species  of  the  organism  and  the  opsonic 
index  changes  obtained  by  their  injection.  In  some  cases  Wright 
has  resorted  to  autoinoculation,  which  is  accomplished  by  mas- 
sage or  manipulation  of  the  infected  part.     This  partly  removes 


OPSONIC    INDEX    FOLLOWING    VACCINE    INJECTIONS.  .")7 

the  toxins  from  the  diseased  tissues  to  healthy  tissues  where  im- 
mune substances  are  actively  produced.  The  amount  of  mas- 
sage and  manipulation  Wright  regulates  by  the  changes  produced 
in  the  opsonic  index. 

While  numerous  investigators  have  reported  results  of  vac- 
cination based  on  opsonic  index  determinations  and  have  ob- 
tained, after  injections,  almost  always  a  uniform  rise  in  the  opsonic 
index,  most  investigators  have  not  found  this  to  be  the  case. 
Even  though  Wright  claims  to  have  obtained  changes  in  opsonic 
index  showing  considerable  regularity,  yet  on  examination  of 
his  charts  one  sees  curves  of  most  marked  irregularity;  the  index 
is  not  constant  in  normal  individuals ;  shows  no  consistent  increase 
or  decrease  in  different  stages  of  infection,  and  is  influenced  by 
many  non-specific  processes  as  menstruation,  exercise,  food,  etc. 
Numerous  investigators  have  found  that  even  though  there  may 
be  first  an  increase  in  opsonic  index  following  vaccination,  yet 
this  is  soon  followed  by  a  fall  of  the  index  and  still  improvement 
of  the  condition  will  be  observed. 

The  results  obtained  on  determinations  of  opsonic  index  in 
health  and  disease  are  so  variable  that  its  importance  as  a  means 
of  diagnosis,  prognosis  and  indication  of  actual  immunity 
must  be  doubted.  It  is  definitely  certain  that  the  index  is  of 
no  value  whatever  unless  carried  out  carefully  by  one  having  had 
considerable  experience  m  its  determination. 


CHAPTER  VI. 


THE  NATURE  OF  OPSONINS 


The  theories  of  Wright  concerning  opsonins  have  received 
attention  chiefly  because  of  the  apparent  value  of  their  quanti- 
tative determination  in  health  and  disease  and  in  the  regulation  of 
dosage  and  interspacing  of  injections  of  bacterial  vaccines.  Nu- 
merotis  investigators,  however,  have  attempted  to  determine  their 
importance  in  immunity,  their  structure,  and  the  conditions 
which  influence  their  action. 

Wright  has  found  opsonins,  the  substances  which  prepare  bac- 
teria for  phagocytosis,  present  in  the  serum  of  normal,  diseased 
and  vaccinated  individuals.  Neufeld  and  Rimpau,  on  the  other 
hand  discovered  substances  which  prepare  bacteria  for  ingestion 
by  leucocytes,  present  in  certain  immune  serum.  Inasmuch  as 
both  the  opsonin  of  Wright  and  the  bacteriotropin  of  Neufeld  and 
Rimpau  perpare  bacteria  for  phagocytosis,  they  are  by  many 
considered  as  identical  substances.  Various  investigators,  while 
admitting  that  these  substances  may  be  identical  in  some  sera, 
do  not  regard  them  as  the  same  substances  in  all  sera.  Wright 
has  regarded  opsonin  as  an  important  factor  in  immunity,  but 
has  made  no  distinction  between  the  opsonin  present  in  normal 
and  immune  sera.  Numerous  investigators,  however,  have  dem- 
onstrated and  emphasized  certain  differences  in  the  bacteriotropic 
substances  in  normal  and  immune  sera. 

SPECIFICITY. 

While  Wright  and  Douglas  did  not  discuss  the  question  of 
specificity  of  opsonins,  still  it  is  evident  that  they  consider  opso- 
nins as  specific.  The  opsonic  index  in  disease,  according  to  Wright, 
shows  certain  deviations  from  the  normal  for  certain  microor- 
ganisms, while  for  other  organisms  it  deviates  but  little  from  the 
opsonic  index  obtained  with  normal  serum.     On  this_]observation 


EFFECT  OF  HEAT  ON  OPSONIN.  59 

Wright  has  based  a  method  for  the  diagnosis  of  certain  microor- 
ganisms in  infections.  This  can,  however,  only  be  regarded  as 
logical  if  opsonins  are  specific.  Bulloch  and  Western  found  that 
in  normal  serum,  by  absorption  tests,  the  specificity  of  opsonin 
for  B.  pyocyaneus,  B.  tuberculosis  and  staphylococci  could  be 
deterinined.  Muir  and  Martin,  Simon,  Russell,  Potter,  Dittman 
and  Bradley,  and  others,  have  found  that  normal  opsonins  are 
not  specific.  Muir  and  Martin,  and  Russell,  however,  by  absorp- 
tion tests  have  determined  that  immune  opsonins  are  quite  spe- 
cific. vSimon,  however,  has  not  been  able  by  investigation  to 
prove  the  existence  of  specific  immune  opsonin. 

EFFECT  OF  HEAT  ON  OPSONIN. 

Wright  has  consistently  assumed  that  normal  and  immune 
opsonins  are  identical  and  has  preferred  to  regard  all  opsonins 
as  thermolabile.  There  are,  however,  on  the  other  hand,  a  large 
group  of  observations  which  show  that  while  opsonins  in  immune 
serum  resist  a  temperature  of  55°  C.  for  one  hour,  opsonins  in 
normal  serum  will  no  longer  be  able  to  prepare  bacteria  for  phag- 
ocytosis by  the  leucocytes  after  such  an  exposure  to  heat.  These 
observations  show  plainly  that  immune  opsonm  is  thermostable, 
while  normal  opsonin  is  thermolabile.  The  basis  for  Wright's 
assumption  that  they  are  all  thermolabile  is  not  evident,  inas- 
much as  Wright  and  Reid  have  shown  that  in  the  serum  of  cer- 
tain patients  suffering  from  tuberculosis  there  is  a  thermostable 
opsonin.  On  this  observation  Wright  has  based  a  method  for 
the  diagnosis  of  tuberculosis,  which  can  only  have  for  a  founda- 
tion the  assumption  that  in  tuberculosis  specific  immune  bodies 
which  are  heat  resisting,  are  produced.  From  the  various  inves- 
tigations reported,  normal  and  immune  opsonins  manifest  a  marked 
dift'erence  in  ability  to  withstand  heat. 

STRUCTURE   OF   OPSONINS. 

Opsonins  have,  by  numerous  investigators,  Savtchenko, 
Besredka,  Loehlein  and  Dean,  been  regarded  as  identical  with 
amboceptors  (fixateur).  Muir  and  Martin  have  shown  that  not 
every  immune  body  produces  an  opsonizing  effect.  Hektoen  has, 
from  a  series  of  experiments,  decided  that  opsonins  are  distinct 
substances   or  anti-bodies.     Neufeld  and   Rimpau,    Neufeld  and 


60  VACCINE    AND    SERUM   THERAPY. 

Toepfer,  Keith,  BuUocli  and  Atkin,  agree  with  Hektoen  and  Wright 
and  Douglas  that  opsonic  action  is  due  to  the  presence  of  hith.erto 
unknown  distinct  bodies. 

Before  this  can  be  accepted,  however,  it  will  be  necessary  to 
repeat  many  of  the  experiments  that  have  been  made,  inasmuch 
as  in  England  and  America  especially,  these  investigations  were 
made  at  a  time  when  no  distinction  was  made  between  normal 
and  immune  opsonins. 

The  existence  of  normal  and  immune  opsonins  is  now  quite 
generally  accepted.  Neufeld  in  a  consideration  of  the  causes  of 
phagocytosis,  states  that  he  believes  bacteria  and  foreign  bodies 
are  only  tkken  up  by  the  leucocytes  when  the  latter  are  stimu- 
lated. He  bases  this  assumption  on  the  phagocytosis  of  red  blood 
cells  by  leucocytes,  which  occurs  only  when  a  special  haemotropic 
serum  is  present.  In  the  haemotropic  serum,  according  to  Neu- 
feld, the  physico-chemical  condition  is  so  changed  that  a  part  of 
the  body  is  modified  so  as  to  serve  as  a  stimulus  or  appetizer  for 
the  phagocytes.  Virulent  organisms  dissolve  with  greater  diffi- 
culty and  give  off  less  appetizer,  and  becatise  of  this  there  is  less 
phagocytosis  of  virulent  organisms  than  of  organisms  with  de- 
creased virulence.  Spontaneous  phagocytosis,  according  to  Neu- 
feld, is  due  to  changes  in  the  cell,  one  of  these  changes  accident- 
ally stimulating  the  leucocytes  to  phagocytosis.  In  immuniza- 
tion Neufeld  believes  a  specific  immune  substance  is  produced, 
which  substance  modifies  bacterial  or  other  cells  so  that  they  will 
serve  to  stimulate  the  leucocytes  to  phagocytosis.  Immune  opso- 
nin or  bacteriotropin,  as  he  prefers  to  call  it,  is  a  thermostable 
substances  which  does  not  require  complement.  Normal  opsonin, 
on  the  other  hand,  is  believed  by  Neufeld  to  produce  its  action 
because  of  normal  amboceptor  and  complement,  which  gently 
dissolves  bacteria  and  in  this  way  stimulates  the  leucocytes  to 
phagocytosis.  The  assumptions  of  Neufeld  are  borne  out  by 
numerous  investigators. 

Immune  opsonins  resist  temperatures  up  to  55°  for  one  hour, 
65°  C.  at  times  not  being  sufficient  to  destroy  their  action.  If 
the  opsonizing  action  of  immune  serum  is  once  lost  it  cannot  be 
regained  by  the  addition  of  fresh  complement.  Muir  and  Martin 
have  found  that  inactivated  immune  opsonin  absorbs  little  or  no 
complement.     Because  of  the  properties  of  immune  opsonins,  they 


STRUCTURE    OF    OPSONINS.  01 

are  generally  regarded  as  belonging  to  the  anti- bodies  of 
the  second  order  of  Ehrlich.  They  apparently  possess  two  groups, 
the  opsonophore  and  the  haptophore.  Of  these  the  opsonophore 
group  is  destroyed  by  heat,  age,  acids,  etc.  It  is  thus  seen  that 
immune  opsonins  resemble  the  agglutinins  and  precipitins  in 
structure  and  by  some  investigators  bave  been  thought  to  be 
identical  with  agglutinins. 

Normal  opsonins,  though  not  acknowledged  by  Wright  to 
be  different  from  immune  opsonins,  have  characteristics  by  which 
they  differ  from  immune  opsonins.  Normal  opsonins  resemble 
complement  in  that  they  are  absorbed  or  fixed  by  sensitized  and 
non-sensitized  bacteria,  blood  corpuscles,  specific  precipitates  and 
indifferent  bodies,  and  exhibit  thermolability  and  susceptibility 
to  deterioration  by  age.  Noguchi  has  found  that  normal  opsonins 
resemble  complement  in  that  they  are  highly  labile  bodies,  loose 
their  action  on  standing  several  days,  are  preserved  for  a  long 
time  when  present  in  blood  m  a  dry  state,  and  in  this  condition 
can  be  heated  to  135°  C.  without  destruction  of  their  functions. 
Recently  Muir  and  Martin,  Levaditi  and  Inman,  and  Huhne  and 
Neufeld  have  ascribed  the  action  of  normal  opsonins  to  comple- 
ment. Cowie  and  Chapin  have  found  that  normal  guinea  pig 
serum  restores  the  opsonic  power  to  normal  serum  which  has  been 
heated  to  55°  C.  They  believe  from  their  experiments  that  opso- 
nins (normal  ?)  exert  their  action  because  of  an  amboceptor-com- 
plement  group.  Hektoen  has  recently  published  results  of  experi- 
ments from  which  he  concludes  that  the  activating  element  is  free 
from  the  opsonin  and  therefore  he  believes  that  opsonins  belong  to 
the  third  order  of  anti-bodies  of  Ehrlich. 

It  now  seems  quite  definitely  established  that  the  action  of 
normal  and  immune  opsonins  is  due  to  entirely  different  factors 
in  immunity,  and  that  immune  opsonins  are  distinct  anti -bodies 
probably  belonging  to  those  of  the  second  order  of  Ehrlich. 

RELATIONS  OF  OPSONINS  TO  LEUCOCYTOSIS. 

No  consistent  relation  has  been  found  to  exist  between  changes 
in  opsonic  index  and  general  or  localized  increase  in  the  number 
of  leucocytes.  Simonds  and  Baldauf  have  recently  found  that  fol- 
lowing an  injection  of  heated  bacteria  there  is  a  decrease  in  leuco- 
cytes after  which  there  is  a  marked  increase  in  leucocytes  with  an 


62  VACCINE    AND    SERUM   THERAPY. 

ultimate  return  to  the  normal.  These  changes  in  the  number  of 
leococytes  they  find  precede  the  changes  in  the  opsonic  index. 
When  the  index  is  at  its  height  the  number  of  leucocytes  has  re- 
turned to  the  normal.  Various  methods  designed  to  increase  the 
number  of  leucocytes  have  been  resorted  to  but  have  all  been  of  little 
avail  in  increasing  the  opsonin  content  in  the  blood  as  determined 
by  Wright's  opsonic  index.  If,  however,  the  leucocytes  can  be 
stimulated  to  phagocytosis  an  increase  in  the  number  of  leucocytes 
in  the  infected  region  must  certainly  be  of  value.  Opsonins  are 
by  some  supposed  to  originate  in  the  leucocytes.  The  importance 
and  possibility  of  this  becomes  more  evident  from  the  work  of 
Peterson  and  Hiss  and  Zinsser.  Potter  believes  that  the  opsonic 
index  varies  with  the  source  of  leucocytes. 

RELATION  OF  OPSONINS  TO  STIMULINS. 

Metchnikoff  has  described  a  series  of  experiments  in  which 
the  introduction  of  serum,  either  from  normal  or  immunized  ani- 
mals, greatly  increased  phagocytosis.  This  action,  he  supposes, 
is  exerted  on  the  leucocytes  and  is  of  great  importance  in  phago- 
cytic immunity.  The  substance  m  the  serum  which  he  regards  as 
stimulating  leucocytes  he  calls  "stimulin."  Stimulins  are  prob- 
ably the  same  as  opsonins,  even  though  up  to  the  present  time, 
in  the  observations  reported,  their  action  is  on  the  leucocytes  and 
not  on  the  bacteria. 


OPSONINS  AND  AGGRESSINS. 

Endotoxins  or  the  aggressins  of  Bail,  have  by  some  been  sup- 
posed to  account  for  decreased  phagocytosis  or  low  opsonic  in- 
dices obtained  with  some  sera.  Aggressins  are  supposed  to  injure 
the  leucocytes  and  inhibit  their  action.  The  part  the  endotoxins 
play  in  preventing  phagocytosis  has  not  yet  been  determined. 
Dorr,  Sauerbek  and  others  have  shown  that  aggressins  are  not 
difinitely  specific.  Neufeld  from  experiments  has  decided  that 
the  lack  of  phagocytosis  with  virulent  organisms  does  not  depend 
upon  the  injury  to  leucocytes  and  for  this  reason  does  not  believe 
that  decreased  phagocytosis  is  due  to  the  action  of  aggressins. 


OPSONINS,    STIMULINS,   AGGRESSINS.  G3 

INFLUENCE  OF  CHEMICALS  AND  REACTION  ON 
•     OPSONINS. 

Various  salts,  even  in  small  amounts,  influence  opsonic  action 
markedly.  Noguchi  has  shown  that  in  normal  serum,  reaction 
exerts  considerable  influence  on  phagocytosis,  it  being  most  marked 
when  the  serum  to  be  tested  is  neutral  in  reaction. 

NON-BACTERIAL  OPSONINS. 

Opsonins  have  been  found  to  be  produced  for  other  cells  than 
bacteria.  Hektoen  has  reported  opsonins  for  blastomycetes  and 
red  blood  cells.  Likewise  it  is  possible  to  get  marked  phagoc5rtosis 
of  inert  particles  of  charcoal,  stains,  etc.,  upon  the  addition  of 
certain  sera. 


CHAPTER  VII. 


VACCINE  THERAPY 


For  centuries  it  has  been  known  that  following  an  attack  of 
certain  acute  infectious  diseases  there  remains  a  certain  loss  of 
susceptibility  to  the  contraction  of  a  second  attack  of  the  same 
disease.  Early  in  the  eighteenth  century  this  experience  was 
utilized  by  vaccination  for  small-pox.  When  it  was  found,  about 
the  middle  of  the  nineteenth  century,  that  many  acute  and  in- 
fectious diseases  have  for  their  etiological  factor  certain  micro- 
organisms known  as  bacteria,  attempts  were  made  to  induce  im- 
munity by  inoculation  of  killed  or  attenuated  cultures  of  these 
organisms.  Pasteur  in  this  way  successfully  immunized  against 
chicken  cholera,  but  while  a  certain  amount  of  success  by  the 
injection  of  killed  cultures  was  obtained,  still  it  was  found  imprac- 
ticable for  reasons  that  are  not  understood  nor  can  be  considered 
here.  Recently,  however,  Haffkine  has  successfully  immunized  or 
vaccinated  against  cholera  and  Pfeiffer  and  Wright  against  ty- 
phoid fever.  It  would  prove  impracticable  to  employ  active  im- 
munization against  all  organisms  with  which  man  may  be  infected, 
for  this  would  require  frequent  injections  of  all  kinds  of  organisms. 
Active  immunization  to  be  applicable  generally  must  be  such  that 
it  can  be  instituted  after  infection  has  occured.  In  this  Pasteur 
was  successful  in  the  disease  of  hydrophobia.  It  was  found,  how- 
ever, that  when  the  method  of  active  immunization  is  begun  after 
infection  has  occured,  there  is  a  superimposition  of  a  mild  form 
of  the  disease  upon  a  severer  form,  thus  accentuating  the  disease. 
This  observation  lead  to  a  relatively  long  period  of  cessation  of 
this  method  of  treatment. 

In  recent  times  active  immunization  after  contraction  of  the 
disease,  has  again  become  important.     This  has  l^een  due  espec- 


BACTERIAL  VACCINES.  65 

ially  to  the  efforts  of  Wright.  It  is  important  to  note  that  the 
method  of  vaccination  was  used  by  Wright  before  he  had  dis- 
covered opsonins.  From  his  later  observations  Wright  has  decid- 
ed that  opsonins  are  of  the  utmost  importance  in  immunity  to 
some  organisms,  and  are  decreased  in  amount  during  most  infect- 
ions but  that  they  can  be  increased  by  the  injection  of  bacterial 
vaccine. 

BACTERIAL  VACCINES. 

It  was  stated  in  a  former  chapter  that  in  bacterial  infections 
the   infected   body   absorbs   bacterial   substances   and   products. 
According  to  the  amount  of  substance  and  products  absorbed 
immune  substances  are  produced.     When  much  is  absorbed  suf- 
ficient immune  substance  is  usually  produced  to  cure  the  disease, 
but  when  little  is  absorbed  not  enough  immune  body  is  produced 
to  cure  the  infection  which  then  becomes  chronic.     To  produce 
enough  immune  substances  to  affect  a  cure  from  an  infection 
Wright  and  Douglas  have  employed  the  well  known  principle  of 
active   immunization.     The   method  they  have   suggested   is  to 
actively  immunize  by  injection  at  the  proper  time,  of  proper  doses 
of  killed  cultures  of  the  organisms  causing  the  infection.     The 
effects  of  injection  of  killed  cultures  or  vaccines  according  to  these 
investigators  is  to  produce  certain  changes  in  the  opsonin  content. 
Wright  and  Douglas  attempt  to  immunize  by  giving  injections 
of  such  numbers  of  bacteria  as  will  produce  only  a  slight  negative 
phase  which  lasts  only  a  short  time  and  is  followed  by  a  rise  in 
opsonic  index  which  lasts  for  some  days.     When  the  index  begins 
to  fall  after  the  subsidence  of  the  positive  phase,  another  injec- 
tion is  made  and  the  effect  on  the  opsonic  index  is  determined. 
An  attempt  is  made  to  inject  such  doses  at  the  second  and  sub- 
sequent injection  that  there  shall  again  be  only  a  slight  negative 
phase  and  that  during  the  positive  phase  the  opsonic  index  shall 
be  higher  than  following  the  first  injection.     An  effort  is  made  to 
keep  the  opsonic  index  of  the  individual  above  normal.     If  the 
index  shows  a  marked  drop  after  any  injection,  the  dose  has  been 
too  large.     By  means  of  vaccines  Wright  claims  that  the  anti- 
bacterial power  of  the  blood  can  be  increased  for  any  microbe 
invading  the  body.     In  1906  Wright  was  able  to  say  that  he  had 
by  this  method  achieved  uniform  success. 


66  VACCINE   AND    SERUM   THERAPY. 

DOSAGE. 

Wright  has,  by  means  of  the  opsonic  index  determined  the 
proper  dosage  for  the  different  bacterial  vaccines  and  while  he  as 
not  stated  that  definite  amounts  must  be  used,  still  he  has  sug- 
gested certain  doses.  His  principle  in  dosage  is  to  use  the  smallest 
dose  that  will  give  a  good  rise  in  the  opsonic  index  and  never  to 
increase  the  amount  injected  until  it  has  been  ascertained  that 
the  doses  which  have  been  injected  have  been  too  small  to  produce 
a  good  rise  in  the  opsonic  index. 

The  doses  of  vaccine  recommended  by  Wright  are  as  follows : 

Bacillus  roli 5  to  50  million  bacteria 

Micrococcus  gonorrhoeae 5  to  50  million  bacteria 

Micrococcus  pneumoniae 10  to  50  million  bacteria 

Bacillus  pyocyaneus 5  to  5  thousand  million  bacteria 

Micrococcus  pj^ogenes 50  to  1  thousand  million  bacteria 

Streptococcus  pyogenes 10  to  25  milHon  bacteria 

Bacillus  tj'hosus 5  to  50  million  bacteria 

Koch's  New  Tubercuhn 1-1,000  to  1-400  milligrams 

These  doses  are  by  some  considered  purely  arbitrary,  because 
the  personal  characteristics  of  the  individual  to  be  immunized, 
the  duration,  extent  and  severity  of  the  infection,  must  all  be  con- 
sidered in  active  immunization.  While  these  doses  may  be  arbi- 
trary they  at  least  offer  some  guide  as  to  the  number  of  organisms 
to  be  injected  at  a  time.  At  times  in  active  immunization  the  in- 
jection of  vaccine  is  accompanied  by  injection  of  specific  immune 
serum.  This  will  pave  the  way  for  larger  doses  and  more  powerftil 
vaccine  injections. 

Injection  of  the  doses  recommended  by  Wright  usually  pro- 
duces no  constitutional  symptoms.  Wright  has  stated  that  at 
times  malaise,  slight  general  muscular  pain  and  headache  are 
observed  on  the  day  following  the  injection  but  as  the  positive 
phase  comes  on  there  is  a  marked  buoyancy  of  spirits  and  stimu- 
lation. Most  investigators  on  this  stibject  have  never  observed 
untoward  symptoms  when  the  small  doses  recommended  by  Wright 
are  injected,  while  others  have  at  times  observed  a  rise  of  tem- 
perature, nausea,  vomiting,  etc.  According  to  the  principle  of 
active  immunization  probably  the  greatest  amount  of  immunity 
results  when  there  is  some  reaction  for  in  active  immunization  the 
individual  or  animal  undergoes  a  modified  or  less  severe  form  of 
the  disease. 


BACTERIAL    A^ACCINE.  67 

The  point  of  injection  of  bacterial  vaccines  is  considered  by 
Wright  to  be  of  importance  in  the  success  of  vaccination.  Those 
regions  of  the  body  are  selected  where  there  is  rapid  absorption 
by  the  lymph  and  blood.  It  has  been  found  that  on  subcutan- 
eous injection  greater  amounts  of  immune  body  are  formed  than 
upon  intravenous  injection.  This  is  especially  illustrated  in 
the  production  of  certain  haemolytic  sera. 

If  Wright's  method  of  vaccine  treatment  of  bacterial  diseases 
is  to  be  judged  it  must  be  remembered  that  the  injection  of  killed 
cultures  did  not  originate  with  Wright,  but  that  Wright  has  added 
opsonic  index  determinations  to  control  the  time  and  doses  of 
these  injections.  Opsonic  index  determinations  have,  however, 
been  found  to  be  open  to  so  many  sources  of  error,  of  such  great 
variability  in  health,  disease  and  after  injections  of  vaccines,  that 
many  clinicians  have  decided  upon  the  dosage  and  inter-spacing 
of  injections  by  clinical  symptoms  rather  than  based  upon  opsonic 
index  determinations.  Most  clinicians  have,  in  the  first  injection,^ 
used  the  dosage  recommended  by  Wright  but  in  subsequent  in- 
jections have  used  such  amounts  as  seemed  warranted  and  indi- 
cated. Hektoen,  adhering  to  the  reliability  of  opsonic  index  de- 
terminations, holds  that  the  index  is  of  diagnostic  and  prognostic 
importance,  while  Park,  Cole,  Bolduan  and  others  regard  the  index 
as  unreliable  for  such  purposes. 

ORGANISMS  USED   IN  VACCINATION. 

Many  of  the  species  of  pathogenic  bacteria  have  been  used  in 
active  immunization.  Two  kinds  of  vaccine  treatment  have  been 
employed,  one  consists  of  the  injection  of  killed  cultures  of  the 
causal  organisms  and  the  other  of  the  autoinoculation  from  the 
infected  focus  by  means  of  massage  and  manipulation. 

In  the  injection  of  vaccines,  so  called  "stock  culture"  vac- 
cines and  "personal"  or  "autogeneous"  vaccines  have  been  used. 
It  has  been  found  that  for  some  organisms,  vaccines  made  from 
stock  cultures  will  give  as  good  results  as  will  personal  or  auto- 
geneous vaccines.  Stock  vaccines  have  the  advantage  that  no 
time  need  be  lost  in  the  preparation  of  the  vaccine,  and  for  nearly 
all  of  the  species  of  bacteria  causing  infections  amenable  to  treat- 
ment by  active  immunization,  stock  vaccines  are  equally  as  good 
as  autogeneous  vaccines.     The  principle  exceptions  are  the  vac- 


68  VACCINE    AND    SKRUM   THERAPY. 

cines  for  Streptococcus  pyogenes,  Mic.  pneumoniae  and,  accord- 
ing to  some,  for  the  Mic.  gonorrhoeae. 

Some  investigators  have  used  vaccines  containing  several 
species  of  bacteria.  Serum  and  vaccine  manufacturers  have  pre- 
pared vaccines  suitable  for  active  immunization  against  one  or 
many  different  sepcies.  Wright  at  one  time  made  no  particular 
effort  to  identify  the  species  of  the  causal  organism,  simply  mak- 
ing a  vaccine  from  the  cultures  obtained  by  inoculation  with 
material  from  the  lesion.  This  is  open  to  man 3^  objections,  for 
at  times  the  causal  organisms  may  be  such  that  it  will  not  grow 
on  ordinary  media  or  else  the  causal  organisms  may  not  grow  as 
well  as  secondary  invaders  and  saprophytic  species.  Because  of 
the  mixed  and  undetermined  vaccines  used  in  immunization  it 
may  frequently  happen  that  those  organisms,  for  which  immuni- 
zation is  necessary  or  beneficial,  are  not  injected. 

In  all  cases  it  ought  clearly  to  be  remembered  that  if  bene- 
fits are  to  be  derived  from  active  immunization  the  vaccine  must 
consist  of  a  suspension  of  the  causal  organism.  To  determine 
these,  microscopic  examinations  of  the  material  from  the  lesion, 
and  isolation  of  the  organisms  when  possible  ought  to  be  made. 
This  cannot  be  too  strongly  emphasized,  especially  to  the  practi- 
tioner who  can  buy  from  the  druggist  vaccines  said  to  be  good 
for  boils,  acne  or  whatever  the  condition  may  be,  while  as  a  mat- 
ter of  fact  the  vaccine  may  not  contain  the  organism  causing  the 
particular  lesion  which  is  to  be  recovered  from  as  a  result  of  active 
immunization.  This  has  undoubtedly  been  the  cause  of  some  of 
the  failures  observed  by  some  clinicians  and  has,  as  was  the  case 
with  tuberculin,  decided  the  practitioner  against  the  use  of  bac- 
terial vaccines  in  certain  cases  of  infection  in  which  vaccine  might 
have  been  of  value. 

PREPARATION  OF  BACTERIAL  VACCINES. 

While  killed  cultures  of  bacteria  have  for  some  time  been 
used  in  active  immunization,  the  amounts  injected  had  been  only 
indefinitely  determined.  Wright  and  Douglas  originated  and  de- 
scribed a  method  for  the  preparation  and  standardization  of  bac- 
terial vaccines  to  be  used  in  immunization.  The  causal  organisms 
according  to  this  method  are  grown  on  artifical  culture  media 
after  which  tliey  are  suspended  in  sterile  salt  solution.    The  num- 


CONTROL  OF  INJECTION  OF  VACCINE.  69 

ber  of  bacteria  per  cubic  centimeter  of  the  suspension  is  deter- 
mined by  drawing  into  a  capillary  pipette  one  volume  of  the 
bacterial  suspension,  and  one  or  more  volumes  of  fresh  blood 
from  a  puncture  in  the  finger.  These  volumes  are  then  mixed 
well  and  a  drop  of  the  mixture  placed  on  a  clean  slide.  With  a 
spreader  which  is  placed  in  front  of  the  drop,  a  smear  is  made. 
This  is  then  allowed  to  dry,  is  fixed  and  stained  with  a  suitable 
stain,  usually  Loeffler's  methylene  blue.  The  slide  is  then  counted 
by  determining  the  number  of  red  blood  cells  and  bacteria  in 
from  ten  to  twenty-five  fields  of  the  high  power  of  the  microscope. 
Normal  human  blood  contains  five  million  red  blood  cells  per 
cubic  millimeter.  After  having  established  the  ratio  of  red  blood 
cells  to  the  bacteria  in  the  suspension,  it  is  easy  to  determine  the 
number  of  bacteria  per  cubic  millimeter  or  cubic  centimeter. 
The  tube  containing  the  bacterial  suspension  is  now  sealed  and 
heated  in  a  water  bath,  to  60  or  65°  C.  for  one  hour.  After  kill- 
ing the  bacteria  in  the  suspension,  a  dilution,  suitable  for  injection, 
is  made  in  a  bottle  containing  50  c.  c.  of  sterile  salt  solution. 
The  bottle  is  closed  with  a  rubber  cap  and  sufficient  carbolic  acid 
or  lysol  is  added  to  make  an  ultimate  dilution  of  0.5  per  cent. 
After  this  the  vaccine  is  tested  for  sterility  on  suitable  culture 
media.  To  obtain  vaccine  from  the  bottle  a  drop  of  pure  lysol 
is  placed  on  the  rubber  cap.  This  is  done  to  sterilize  the  outer 
surface  of  the  rubber  cap.  Then  the  sterile  hypodermic  needle 
is  inserted  through  the  cap,  the  bottle  turned  up  and  the  piston 
of  the  syringe  is  withdrawn  until  the  desired  amount  of  vaccine 
has  been  taken  into  the  barrel  of  the  syringe. 

CONTROL  OF  INJECTION. 

While  Wright  and  his  pupils  have  claimed  to  regulate  the 
dosage  and  inter-spacing  of  injections  entirely  by  the  opsonic 
index,  others  have  found  the  opsonic  index  too  unreliable  to  serve 
as  a  guide  in  the  administration  of  bacterial  vaccines.  Wright 
and  his  pupils  have  not  always  based  their  decisions  on  dosage 
and  time  for  injection,  on  determinations  of  the  opsonic  index. 
It  frequently  happens  that  at  the  time  of  the  first  injection  of 
vaccine  their  patients  have  an  opsonic  index  well  above  normal. 
In  most  cases  the  treatment  is  based  on  determinations  made  by 
serum  drawn  twenty-four  hours  earlier.     The  time  for  the  second 


70  VACCINE    AND    SERUM    THERAPY. 

and  subsequent  injection  is  not  always  controlled,  even  by  Wright 
and  his  pupils,  by  the  opsonic  index. 

In  accepting  opsonins  as  important  factors  in  immunity  other 
immune  substances  must  not  be  lost  sio^ht  of.  Various  investi- 
gators  have  designed  methods  by  which  the  action  of  agglutinins 
and  bacteriolysins  is  to  be  inhibited,  so  that  opsonic  indices  may 
be  determined  and  used  as  a  guide  for  the  dosage  and  inter-spac- 
ing of  injections.  The  justifications  for  such  regulation  of  dosage 
and  determination  of  the  proper  time  for  injection  is  not  evident. 
In  a  patient  suffering  with  cerebro-spinal  meningitis,  from  the 
spinal  fluid  of  which  Mic.  meningitidis  was  cultivated,  the  writer 
tried  to  govern  the  dosage  and  inter-spacing  of  injections  of 
meningococcus  vaccine  by  the  opsonic  index.  After  three  to 
four  minutes  of  incubation  of  the  mixture  of  leucocytes,  men- 
ingococcus suspension  and  patient's  serum,  all  of  the  organisms 
w^ere  found  to  be  dissolved.  It  is  evident  that  lysins  in  this  case 
were  probably  of  more  importance  in  immunity  than  were  opso- 
nins. Clark  has  proposed  that  in  determining  the  opsonic  index 
of  serum  of  typhoid  patients,  the  serum  be  heated  to  56°  C.  in 
order  to  destroy  the  lysins  for  the  typhoid  bacillus.  By  follow- 
ing out  this  method  Clark  finds  that  relapses  follow  upon  a  drop 
in  the  opsonic  index.  How  much  more  important  would  it  prob- 
ably be  to  determine  the  curve  for  lysins  in  a  case  where  lysins 
are  present  in  such  amount  that  it  is  necessary  to  destroy  their 
action  before  opsonic  index  determ'nations  can  oe  made?  This 
would  seem  more  rational,  especially  because  our  knowledge  con- 
cerning lysins  in  immunity  is  greater  than  it  is  concerning  opsonins, 
which  may  or  may  not  play  any  part  in  immunity. 

According  to  the  determinations  of  the  opsonin  content  of 
the  blood,  Wright  and  his  pupils  have  found  that  the  opsonic 
index  begins  to  rise  two  to  five  days  after  injection,  and  usually 
reaches  its  height  after  about  five  to  eight  days.  In  most  cases 
adherents  of  the  opsonin  theory  make  injections  every  four  to 
eight  days.  The  appearance  in  the  blood,  however,  of  the  better 
known  anti-bodies  comes  somewhat  later.  Bacteriolysins  are 
found  to  be  present  at  times  in  the  spleen  twenty-four  hours  after 
injection  of  culture.  They  do  not,  however,  appear  in  quantities 
in  the  blood  until  five  to  nine  or  fourteen  days  later.  Agglutinins 
are  found  to  be  present  in  the  blood  from  eight  to  twelve  days 


ANTI-BODIES   AFTER    VACCINE    INJECTIONvS.  71 

after  injection  of  cultures.  Typhoid  agglutinins  ""usually  do  not 
appear  in  a  typhoid  patient's  blood  until  after  ten'^days  of  the 
disease  have  elapsed.  Furthermore,  it  is  noted  that  anti-toxins, 
bactericidins  and  agglutinins  can  be  enormously  increased  by 
correct  inter-spacing  of  injections  and  proper  dosage.  Wright 
himself  has  called  attention  to  the  value  of  determining  these 
different  substances  in  immunity  before  injections  are  repeated. 
If  injections  are  made  entirely  according  to  the  opsonic  index,  they 
may  be  made  before  the  bactericidal  and  other  protective  sub- 
stances have  increased  in  amount,  several  negative  phases  may 
thus  be  superimposed  on  each  other,  and  the  protective  substances 
be  mtich  decreased.  With  the  role  of  opsonins  in  immunity  in 
doubt,  the  difference  in  sensibility  of  different  species  of  microor- 
ganisms to  opsonins  established,  and  the  presence  of  marked 
bacteriolytic  action  of  certian  sera  for  certain  species  of  infecting 
organisms  observed,  the  control  of  the  process  of  active  immuni- 
zation by  the  opsonic  index  alone  does  not  seem  justifiable.  The 
immune  bodies  of  importance  in  immunity  to  the  particular  species 
of  bacteria  ought  to  be  established  so  that  the  dosage  and  inter- 
spacing of  injections  could  be  regulated  by  determinations  of  the 
presence  of  these  substances.  Meakins  has,  on  this  basis,  studied 
immunity  to  dysentery  bacilli.  Streptococcus  pyogenes,  Micro- 
coccus pyogenes  and  Bacillus  tuberculosis.  Such  methods  would 
undoubtedly  result  in  a  better  accomplishment  of  a  higher  grade 
of  active  immunity. 

It  is  necessary,  however,  to  refer  to  the  great  success  obtained 
by  Wright,  and  others  following  Wright's  teachings,  in  the  treat- 
ment of  bacterial  infections  and  diseases  according  to  the  deter- 
minations of  the  opsonic  index.  Wright  has  stated  that  he  has 
not  achieved  success  only  in  a  certain  percentage  of  cases,  but  that 
he  has  had  uniform  success  by  following  his  method  of  active  im- 
munization. Ross  has  given  a  list  of  diseases  successfully  treated 
based  on  the  determinations  of  the  opsonic  index.  This  list  in- 
cludes furunculosis,  pustular  acne,  sycosis,  cystitis,  otitis  media, 
empyema,  gleet,  gonorrhoeal  rheumatism,  acute  gonorrhoea,  local- 
ized tuberculosis,  lupus,  tuberculosis  of  the  joints,  bladder,  kid- 
ney, epididymis,  glands,  peritoneum,  iris  and  lungs.  Surely  a 
method  that  will  eive  uniform  success  for  as  large  a  list  of  dis- 


72  VACCINE    AND    SERUM    THERAPY. 

eases  as  this,  is  one  tliat  must  be  of  tlie  utmost  importance  in 
clinical  medicine. 

Because  the  methods  for  the  determination  of  immune  bodies 
are  still  so  inaccurate  and  in  many  cases  unreliable,  and  because 
the  defensive  agencies  of  the  animal  body  against  bacterial  in- 
vasion and  multiplication  are  so  manifold  and  complex,  numer- 
ous investigators  have  relied  upon  the  clinical  control  of  dosage 
and  inter-spacing  of  injections  of  bacterial  vaccine.  At  all  events, 
the  value  of  active  immunization  by  injection  of  bacterial  vaccines 
will  ultimately  rest  upon  clinical  results.  While  it  is  relatively 
easy  in  most  cases  of  infection  to  tell  when  complete  recovery 
has  taken  place,  still  it  is  difficult  to  tell  what  clinical  symptoms 
and  signs  are  associated  with  the  best  method  of  immunization. 
The  conditions  ultimately  desired  are  definitely  known,  but  the 
conditions  in  the  diseased  body  which  will  produce  these  ulti- 
mately desired  effects,  are  little  understood,  Probably  the  best 
illustration  of  this  is  seen  in  the  use  of  tuberculin  in  the  treatment  of 
tuberculosis.  Tuberculin  has  been  known  and  used  in  treatment 
for  nearly  twenty  years,  and  still  today  it  is  an  open  question 
whether  the  dosage  shall  be  regulated  to  give  an  anti-toxic  or  an 
anti -bacterial  immunity.  Clinical  evidence  and  experience  is 
deceiving  as  is  well  demonstrated  in  the  literature  on  tuberculin. 

With  some  organisms,  and  in  certain  cases  treated  by  the 
vaccine  method,  excellent  results  have  been  obtained  when  the 
dosage  and  interval  between  injections  has  been  determined  by 
clinical  signs  and  symptoms.  The  number  of  cases  in  which 
the  treatment  is  controlled  clinically  is  probably  larger  than  the 
number  of  those  treated  according  to  Wright's  method.  Many 
investigators,  although  determining  the  opsonic  index  during  the 
process  of  immunization,  have  found  that  it  is  quite  as  safe  to 
administer  vaccine  when  controlled  by  clinical  results  as  by  op- 
sonic index  determinations.  In  clinical  control  of  vaccine  treat- 
ment, it  is  aimed  to  have  the  dose  so  small  and  the  injections  so 
far  apart  that  immunization  is  accomplished  without  the  pro- 
duction of  clinical  symptoms.  The  doses  that  are  usually  given 
are  those  recommended  by  Wright,  though  some  have  used  larger 
doses  without  the  production  of  any  reaction. 

In  most  clinics  the  doses  injected  are  gradually  increased. 
This   is   also   the   procedure   recommended   by   Wright.     If   this 


STAPHYLOCOCCUS    VACCINE.  73 

method  is  followed,  it  frequently  happens  that  there  may  be  an 
exacerbation  in  the  pathological  condition.  This  is  especially 
observed  in  furunculosis  produced  by  the  staphylococci.  Fre- 
quently following  the  second  injection  of  a  vaccine  of  this  organ- 
ism a  new  furuncle  will  appear.  The  writer  has  obtained  better 
results  by  first  injecting  the  largest  dose  and  then  at  subsequent 
injections  to  give  constantly  decreasing  doses.  The  reasons  for 
following  this  method  are  based  entirely  upon  clinical  experience 
and  may  be  due  to  the  fact  that  if  the  second  and  subsequent 
injections  are  larger  than  the  preceding  one,  too  much  of  the  im- 
mune substance  is  used  up  in  combining  with  the  bacterial  vac- 
cine injected  or  there  may  be  an  actual  hypersusceptibility  to 
these  organisms. 

The  interval  between  injections  when  the  opsonic  index  is 
not  determined,  lies  usually  between  seven  and  ten  days,  which 
is  about  the  time  required  for  the  production  of  known  immune 
substances.  Whenever  a  reaction  follows  an  injection  the  in- 
terval between  injections  is  lengthened  so  as  to  allow  sufficient 
time  for  the  disappearance  of  all  symptoms. 


Staphylococcus  Infections.  The  various  infections  caused  by 
the  Micrococcus  pyogenes  group  have  been  considered  most  satis- 
factory for  vaccine  treatment.  In  the  list  of  staphylococcus  infec- 
tions treated  by  bacterial  vaccines,  Ross  includes  boils,  carbuncles, 
acne,  sycosis,  felons,  styes,  and  septic  wounds.  The  results  ob- 
tained in  the  treatment  of  furuncles  and  carbuncles  by  killed 
cultures  of  the  infecting  organisms  have  varied.  Probably  the 
best  results  have  been  obtained  in  localized  surface  infections. 
In  the  treatment  of  these  cases  it  has  been  found  that  autogen- 
ous vaccines  are  not  necessary.  It  is,  however,  important  in  all 
cases  to  determine  the  species  and  variety  of  organisms  causing 
the  infection  and  to  use  this  variety  in  the  treatment  of  the  path- 
logical  conditions.  The  staphylococcus  vaccines  with  which  the 
writer  has  obtained  the  best  results  are  those  made  from  different 
isolation  of  the  same  variety  of  organisms.  Thus  a  Mic.  pyogenes 
aureus  vaccine  which  has  given  good  results  is  one  made  from 


74  VACCIXK    AND    SERUM    THERAPY. 

three  isolations,  the  organisms  being  isolated  from  the  lesions  of 
chronic  marked  furunculosis,  from  an  acute  boil  on  the  face,  and 
from  a  patient  having  multiple  pustules  on  the  back  of  the  neck. 
Most  patients  suffering  with  fumucles  and  carbuncles  show  a 
marked  improvement  within  twenty-four  hours  after  injection 
and  some  of  these  patients  remain  well  after  that.  Everyone 
who  has  treated  a  number  of  patients  suffering  with  furuncles  or 
carbuncles  must  have  observed  that  there  occasionally  is  recur- 
rence of  these  conditions  after  apparent  recovery  following  upon 
the  injection  of  suitable  vaccines.  If  Wright's  method  of  vacci- 
nation according  to  the  opsonic  index  is  followed  out,  there^  are 
apparently  more  recurrences  than  w^hen  the  injections  are  given 
at  intervals  not  less  than  seven  days  nor  greater  than  ten  days. 
In  all  cases  the  best  results  will  be  obtained  if  a  small  incision  is 
made  in  the  carbuncle  or  furuncle  twenty-four  hours  after  vacci- 
nation. By  this  method  the  collection  of  leucoc3rtes  or  pus  is 
drawm  off,  the  pressure  is  relieved  and  the  bacteria  ingested  by 
the  leucocytes  are  removed.  The  advantage  of  this  becomes 
evident  when  it  is  realized  that  staphylococci  secrete  a  leucocidin, 
which  destroys  leucocytes.  If  the  leucocytes  containing  bacteria 
can  be  removed  before  they  are  killed  the  value  of  phagocytosis 
in  immunity  is  evident.  After  this  the  pus  is  evacuated  daily 
from  the  furuncle  or  carbuncle  and  the  wound  is  not  allowed  to 
heal  until  it  has  been  found  that  for  several  days  no  more  pus  is 
being  formed.  In  addition  to  draining  the  furuncle  or  carbuncle 
through  a  slight  incision,  thorough  cleansing  of  the  skin  with 
benzine  is  of  great  advantage  in  preventing  the  formation  of  new 
foci.  The  doses  used  in  most  clinics  for  these  conditions  are  gradu- 
ally increased,  the  first  injections  usually  consisting  of  three 
hundred  million  cocci.  As  was  mentioned  before,  apparently 
better  results  are  obtained  when  at  the  first  injection  six  hundred 
million  cocci  constitute  the  dose,  while  the  later  doses  contain 
fewer  cocci,  at  times  only  one  hundred  million.  While  this  method 
of  vaccination  does  not  depend  for  its  results  only  on  the  effects 
of  vaccines,  still  it  is  a  method  which  gives  good  results  without 
the  formation  of  large  scars. 

Acne  vulgaris  has  been  successfully  treated  by  vaccination 
only  in  certain  cases.  The  value  of  bacterial  injections  in  this 
condition   was   so  emphasized  by  Wright  that  for  a  time  it  was 


STREPTOCOCCUS    VACCINE.  75 

the  common  belief  among  clinicians  that  vaccinations  with  killed 
cultures  of  Micrococcus  pyogenes  albus  were  applicable  in  all  cases 
of  acne.  This,  however,  is  not  the  case.  In  the  lesions  of  many 
patients  suffering  with  acne  vulgaris,  the  white  vskin  coccus  cannot 
be  found.  In  such  cases  either  the  species  of  organisms  causing 
the  pustules  must  be  injected  or  else  vaccine  treatment  should 
not  be  applied.  A  number  of  the  cases  of  acne  which  have  been 
treated  by  the  writer  have  failed  to  show  microorganisms  that 
could  be  cultivated.  When  the  vaccine  treatment  is  used  other 
means  of  treatinent  must  not  be  discontinued.  Incisions  and 
evacuation  of  the  pustules,  cleansing  of  the  skin  with  benzine, 
expression  of  comedones  or  black  heads,  the  bathing  of  the  infected 
part  in  hot  water,  application  of  sulphur  or  other  suitable  lotions 
and  the  regulation  of  the  diet  are  all  of  assistance  to  overcome 
this  disease.  Even  under  these  measures  the  treatment  is  not 
always  successful. 

The  treatment  of  sycosis  barbae  or  barber's  itch  with  staphy- 
lococcus vaccines  has  not  always  been  as  successful  as  has  been 
reported.  Two  patients  with  this  disease  in  the  City  Hospital 
in  New  York  City,  were  treated  by  Ross  with  injections  of  staphy- 
lococcus vaccine.  No  satisfactory  results,  however,  were  obtained. 
The  unsatisfactory  results  obtained  by  the  injection  of  staphy- 
lococcus vaccine  is  easily  explained  when  it  is  realized  that  sta- 
phylococci cause  secondary  infections  in  only  some  of  the  cases 
of  sycosis  barbas.  In  these  cases  only  can  staphylococcus  vaccine 
be  of  benefit. 

Streptococcus  Infections.  Various  kinds  of  streptococcus  in- 
fections have  been  treated  by  vaccines.  Usually  these  infections 
are  acute  and  severe  and  it  often  is  a  question  whether  sufficient 
time  for  the  production  of  immune  bodies  as  a  result  of  vaccina- 
tion will  elapse  before  death  or  spontaneous  recovery  terminates 
the  disease.  Arthrites,  pneumonia,  pericarditis,  erysipelas,  local 
infections  and  endocarditis,  etc.,  have  been  treated  by  the  use  of 
vaccine.  At  times  striking  results  have  been  obtained,  while 
in  other  cases  not  treated  by  vaccination  there  has  been  equally 
striking  recovery.  One  needs  only  to  remember  the  cases  of 
streptococcus  infections  of  the  uterus,  of  septicaemia  and  ery- 
sipelas that  recover  in  a  few  days  without  any  specific 
treatment,  to  realize  that  it  is  easy  to  attribute  beneficial  results 


76  VACCINE    AND    SERUM   THERAPY. 

to  the  use  of  streptococcus  vaccines  in  cases  in  which  the  same 
results  might  have  been  obtained  without  them.  In  1907,  the 
author  treated  37  patients  suffering  with  erysipelas  by  injections  of 
killed  cultures  of  streptococci  isolated  from  the  lesions  of  ery- 
sipelas. From  the  results  obtained  it  was  impossible  to  determine 
the  value  of  the  injection  of  the  killed  cultures  of  streptococci. 
While  the  injection  of  from  twenty-five  million  to  one  hundred 
million  streptococci  did  not  prevent  migration  and  recurrence, 
the  apparent  shortening  of  the  duration  of  the  disease  suggests 
that  streptococcus  vaccines  are  of  some  value.  The  effect  upon 
the  duration  of  the  disease  was  found  to  be  uncertain  because, 
during  the  time  of  the  investigation,  no  suitable  cases  remained 
untreated  with  which  comparison  could  be  made.  It  is  known  that 
the  duration  of  the  disease  varies  in  different  j^ears.  Ross  and 
Johnson  have  recently  reported  on  the  treatment  of  erysipelas 
with  a  vaccine  made  from  streptococcus  erysipelatos,  and  conclude 
that  such  vaccine  exercises  a  specific  and  controlling  influence  on 
the  course  of  the  disease.  These  investigators  make  initial  injec- 
tions of  from  ten  to  twenty  million  killed  streptococci,  and  repeat 
these  injections  every  day  or  two  according  to  the  clinical  results 
obtained.  Criticism  of  the  results  obtained  is  not  possible  because 
of  the  meagre  reports  of  the  cases  treated  by  these  investigators. 

Wright  and  others  have  reported  successful  treatment  of 
streptococcus  septicaemia  by  the  injection  of  streptococcus  vac- 
cine. It  must,  however,  be  remembered  that  streptococcus  septi- 
casmia  is  at  times  recovered  from  even  when  no  specific  treatment 
is  given. 

In  streptococcus  immunization  autogenous  vaccines  have 
usually  been  preferred,  though  Gabritschewsky  and  others  have 
gotten  good  results  with  stock  vaccines. 

Gonococcus  Infections.  Probably  the  best  results  of  the  use 
of  gonococcus  vaccines  have  been  obtained  in  cases  of  gonorrhoeal 
arthritis.  Cole  and  Meakins  obtained  encouraging  results  in 
about  twenty  cases  of  acute  and  chronic  gonorrhoeal  arthritides 
when  treated  by  injections  of  stock  vaccines  of  Mi c.  gonorrhoeae. 
Tliese  investigators  treated  their  cases  according  to  the  opsonin 
content  of  the  blood  as  determined  by  the  opsonic  index,  but 
feel  that  probably  as  good  results  might  have  been  obtained  had 
the  injection  been  given  every  ten  days,   which  was  about  the 


GONOCOCCUS   AND    PNEUMOCOCCUS    VACCINE.  77 

time  determined  upon  by  the  opsonic  index.  The  doses  injected 
in  these  cases  varied  from  five  hundred  million  to  one  thousand 
million  cocci. 

Treatment  of  acute  and  chronic  urethritis  by  vaccines  has 
given  some,  but  relatively  few,  beneficial  results,  and  is  by  no 
means  to  be  regarded  as  a  sufficient  measure  to  obtain  a  cure  from 
t  his  condition. 

Micrococci  pneumoniae  Infections.  Apparently  some  good  re- 
sults have  been  obtained  in  the  treatment  of  chronic  infections 
with  this  organism.  The  dose  injected  varies  from  ten  million  to 
one  hundred  million.  In  acute  pneumonia  the  course  of  the 
disease  is  usually  too  short  to  derive  beneficial  results  by  means 
of  active  immunization. 

Tuberculosis.  The  bacillus  of  tuberculosis  may  produce  its 
lesions  in  almost  any  part  of  the  body.  Bacteriological  examina- 
tion of  the  lesions  produced  will  vary.  In  some  lesions,  especially 
those  localized  in  the  interior  of  the  body  such  as  the  glands, 
joints  and  bones,  the  tubercle  bacillus  alone  is  responsible  for  the 
condition  found.  In  other  lesions,  as  in  pulmonary  and  intestinal 
tuberculosis,  there  are  present  in  addition  to  tubercle  bacilli  other 
bacterial  species.  x\ctive  immunization,  by  the  injection  of  killed 
tubercle  bacilli  theroetically  will  be  of  benefit  in  lesions  due  to 
the  tubercle  bacillus  alone,  while  in  lesions  produced  by  tubercle 
bacilli  and  other  species  it  will  not  be  very  efficacious. 

In  the  specific  treatment  of  tuberculous  conditions,  various 
modifications  of  so-called  tuberculin  have  been  used.  Old  tuber- 
culin is  the  concentrated  germ-free  glycerine  bouillon  on  which 
tubercle  bacilli  have  been  grown  for  several  weeks.  Before  fil- 
tration the  tubercle  bacilli  are  killed  by  heat.  Deny's  bouillon 
filtrate,  "B.  F."  differ  from  old  tuberculin  in  that  filtration  is  done 
before  killing  the  bacteria  by  heat.  Tuberculin  R,"T.R."  con- 
tains the  powdered  tubercle  bacilli  suspended  in  salt  solution. 
Usually  1  c.  c.  contains  2  m.  g.  of  powdered  bacilli.  Tuberculin 
0,  "T.  0.*'  is  the  supematent  opalescent  liquid  obtained  after 
emulsifications  of  ground  up  tubercle  bacilli  in  distilled  water, 
are  centrifuged.  Bacillen  Emulsion,  *'B.  E."  is  a  suspension  of 
ground  up  tubercle  bacilli  in  fifty  per  cent  glycerine.  It  contains 
5  m.  g.  in  1  c.  c.  of  suspension. 


78  VACCINE   AND    SERUM   THERAPY. 

Our  knowledge  concerning  the  effect  of  tuberculin  on  the 
bacillus  of  tuberciilosis  and  the  tissues  of  the  body  in  which  the 
bacillus  is  found,  is  still  indefinite.  Before  tuberculin  can  be  scien- 
tifically applied  as  a  therapeutic  measure,  it  is  necessary  to  deter- 
mine the  part  played  by  the  moderate  reaction  in  the  tissues  and 
what  is  to  be  accomplished  by  immunization  with  tuberculin. 

The  reaction  in  diseased  tissues  following  the  injection  of 
tuberculin  may  be  either  mild,  in  which  case,  there  is  redness  due 
to  hyperaemia  and  congestion,  or  it  may  be  marked,  producing 
necrosis  and  sloughing  of  the  diseased  tissues.  In  all  attempts 
at  immunization  with  tuberculin  the  mild  reaction  is  preferred 
because  it  does  not  lead  to  the  liberation  of  tubercle  bacilli  as  a 
result  of  the  necrosis  of  tissues,  but  does  furnish  to  the  lesions 
an  increased  blood  supply. 

The  conception  of  the  immunization  accomplished  by  tuber- 
culin injections  differs,  there  being  two  important  theories.  Koch, 
Wright  and  others  believe  that  anti-body,  specific  to  the  action 
of  the  tubercle  bacillus,  is  produced.  This,  according  to  their 
views,  is  accomplished  through  stimulation  of  all  the  defenses 
of  the  body.  In  conformity  to  their  views  on  the  objects  of 
tuberculin  immunization,  tubercle  bacilli  are  injected  in  the  form 
of  "T.  R."  or  "B.  E." 

Maragliano,  Sahli,  Denys,  Trudeau,  and  others  adhere  to  the 
toxin  immunization  theory,  according  to  which  tuberculin  injec- 
tions lead  only  to  tolerance  or  immunization  to  the  toxin  liberated 
by  the  tubercle  bacillus.  In  accordance  to  this  theory,  old  tuber- 
culin as  well  as  other  tuberculins  are  injected,  the  doses  being 
gradually  increased  to  the  highest  point  of  tolerance.  The  lesion 
according  to  this  theory  is  only  secondarily  effected,  there  being 
no  acquisition  of  immunity  to  the  tubercle  bacillus. 

Tuberculin  treatment  is  usually  only  undertalvcn  in  tuber- 
culosis of  the  glands,  joints  and  bones,  though  in  some  clinics  it 
has  also  been  used  in  pulmonary  and  other  forms  of  tuberculosis. 
Koch  would  limit  its  application  to  such  cases  as  are  still 
curable — -that  is,  have  not  advanced  too  far  nor  are  complicated 
by  other  bacteria.  In  deciding  upon  cases  amenable  to  tuber- 
culin treatment  Koch  relies  upon  the  body  temperature  as  the  indi- 
cator. In  uncomplicated  and  curable  cases  the  temperature  does  not 
rise  mtich  above  normal.     Various  measures  liave  been  taken  as 


TUBERCULINE,    COLON   AND   TYPHOID    BACILLUS    VACCINE.  7'.) 

guides  in  tuberculin  treatment  of  tuberculosis.  Wright  injects 
only  amounts  large  enough  to  produce  a  positive  phase  in  the 
opsonic  index  and  in  some  cases  of  tuberculous  glands  and  joints 
has  accomplished  good  results  by  autoinoculation.  Trudeau, 
Denys  and  others  have  regulated  the  dosage  and  inter-spacing  of 
injections  by  clinical  signs  and  symptoms.  They  usually  inject 
1-1000  m.  g.  of  old  or  1-10,000  m.  g.  of  "Bacillen  Emulsion." 
After  this,  by  a  long  continued  treatment  they  gradually  increase 
the  dosage.  The  doses  injected  are  not  increased  nor  the  interval 
between  injections  shortened  if  there  are  any  general  or  local  re- 
actions, as  evidenced  by  fever,  local  redness  and  edema.  In  this 
method  no  stress  is  laid  on  the  absolute  amount  of  tuberculin 
injected.  When  the  patient  has  no  tolerance  to  tuberculin,  Tru- 
dean  has  usually  observed  chronic  toxaemia  and  progression  of 
the  disease. 

Tuberculin  treatment  has  proven  especially  beneficial  in 
lesions  in  the  joints,  bones,  glands,  skin  and  urinary  bladder, 
although  good  results  have  been  obtained  in  pulmonary  and  other 
formis  of  tuberculosis. 

Bacillus  coli  Infections.  Different  infections  produced  by 
Bacillus  coli  have  been  treated  by  injections  of  vaccines  made  with 
this  organism.  The  vaccine  method  of  treatment  has  been  found 
to  be  most  successful  in  cases  of  chronic  cystitis  due  to  B.  coli  com- 
munis, although  cholecystitis,  appendix  abscesses,  endometritis 
and  other  local  infections  with  this  organism,  have  at  times  re- 
sponded favorably  to  this  method  of  treatment.  The  number  of 
bacteria  injected  is  seldom  more  than  fifty  million,  usually  smaller 
doses  than  this  being  used.  Vaccines  made  from  cultures  isolated 
from  the  patient  usually  give  the  best  results.  The  injections  are 
repeated  about  every  ten  days. 

Bacillus  typhosus  and  Bacillus  paratyphosus  Infections.  The 
typhoid  bacillus  group  consist  of  several  important  members. 
Recognition  of  this  fact  has  led  to  the  use  of  vaccine  made  from 
cultures  isolated  from  the  patient  to  be  treated.  Vaccines  made 
from  killed  cultures  of  typhoid  bacilli  have  been  used  for  immuniz- 
ation as  well  as  for  treatment  of  pathological  conditions  due  to 
members  of  this  group. 

Pfeiffer  and  Kolle  have  made  vaccines  for  immunization 
against  typhoid  fever  from  agar  cultures,  while  in  England  the 


80  VACCINE    AXD    SERUM    THERAPY. 

vaccine  usually  is  made  from  bouillon  cultures.  The  organisms 
are  killed  by  one  hour's  exposure  to  53°  C.  to  60°  C.  Pfeiffer  and 
Kolle  inject  sufficient  amounts  of  vaccines  to  produce  a  local  reac- 
tion lasting  several  days,  and  a  general  reaction,  as  is  demon- 
strated by  fever  and  malaise,  lasting  one  to  two  days.  Following 
the  first  injection,  two  to  three  injections  of  increasing  amounts 
are  made  at  intervals  of  eight  days.  The  English  method  also 
produces  marked  disturbances  at  times.  The  duration  of  immunity 
confen-ed  by  this  method  of  treatment  varies,  and  is  usually  re- 
garded as  lasting  from  one  to  three  years.  The  results  of  such  im- 
munization are  still  doubtful  but  statistics  indicate  that  immunized 
individuals  are  less  likely  to  contract  the  disease,  and  that  when 
the  disease  is  contracted  it  is  less  severe  and  less  often  fatal. 

Typhoid  bacillus  vaccine  has  been  tried  in  the  treatment  of 
typhoid  fever.  Here  it  has  met  with  but  little  success.  The  best 
results  with  the  vaccine  have  been  obtained  in  cystitis,  cholecysti- 
tis, and  the  local  abscesses  following  attacks  of  typhoid  fever.  The 
doses  injected  range  usually  from  five  million  to  fifty  million  bacilli, 
the  injections  being  repeated  at  intervals  from  eight  to  ten  days. 
Autogenous  A^accines  usually  give  the  best  results. 

Bacillus  dysenteriae  Infections.  Vaccines  made  from  dysen- 
tery bacilli  have  been  used  in  prophylaxis  as  well  as  treatment  of 
bacillary  dysentery.  Inasmuch  as  there  are  different  strains  of 
these  bacilli,  it  is  advisable  to  use  vaccines  made  from  the  cultures 
isolated  from  the  patient  or  with  all  of  the  different  strains  of  B. 
dysenteriae. 

Shiga  has  tried  various  methods  of  active  immunization  and 
has  foimd  that  the  best  results  are  obtained  either,  by  injecting 
subcutaneously  anti-dysenteric  serum  and  killed  cultures  of  dysen- 
tery bacilli,  or,  by  taking  by  mouth  the  killed  cultures  of  this  organ- 
ism. By  Shiga's  m.ethod  of  simultaneous  injection  of  anti-dysen- 
teric serum  and  dysentery  vaccine  the  death  rate  in  Japan  due  to 
bacillary  dysentery  has  been  much  reduced.  The  duration  of  im- 
munity, according  to  Shiga,  is  about  two  months. 

Vaccine  treatment  of  d37-sentery  can  only  be  applied  in  cer- 
tain cases.  By  the  method  of  active  immunization,  immunity  can- 
not be  established  in  much  less  than  eight  days,  and  for  this  reason 
only  subacute  or  chronic  cases  are  amenable  to  treatment  by  vac- 
cines.    The  numiber  of  cases  .treated  by  this  method  is  still  too 


IMMUXIZATIOX    AGAINST   HYDROPHOBIA.  81 

small  to  warrant  a  conclusion.  It  seems  quite  certain,  however, 
that  vaccine  therap}^  may  prove  of  value  in  this  disease.  It  must 
be  borne  in  mind  that  there  are  different  strains  which  can  produce 
the  disease  and  that  an  autogenous  vaccine  will  most  likely  give 
the  best  results.  Usually  fifty  million  bacilli  are  injected,  the  inter- 
val between  injections  being  from  eight  to  ten  days. 

Cholera  Vaccines.  Vaccines  made  from  cholera  vibrions  have 
been  used  in  protective  immunization.  Statistics  on  this  method 
are  still  too  meagre  to  warrant  definite  conclusions  on  its  value. 

Less  common  Infections.  Vaccine  treatment  has  been  ap- 
plied in  most  all  infections  produced  by  bacteria.  The  results 
obtained  have  varied,  though  Wright  believes  that  the  method 
is  applicable  in  the  treatment  of  all  infections  caused  by  bacteria. 

SPECIFIC  VACCINES  FOR  DISEASES  OF 
UNKNOWN  ETIOLOGY. 

Rabies,  Hydrophobia  or  Lyssa.  This  is  a  disease  whose  eti- 
ology is  only  indefinitely  established.  The  organism  responsible 
for  the  condition  has  never  been  crrown.  Bv  some  investigators, 
certain  peculiar  bodies,  found  in  1903  by  Negri  in  the  large  nerve 
cells  in  the  central  nervous  system,  are  regarded  as  the  causal  factors 
in  the  disease.  Although  these  bodies  are  not  iniiversally  accepted 
as  the  etiological  factors,  they  are  quite  generally  accepted  as  spe- 
cific to  this  disease. 

The  inability  to  establish  the  etiolog}^  has  not  prevented  the 
establishment  of  methods  of  immunization  to  this  disease.  The 
principle  of  the  method  of  immunization  most  generally  practised 
is  based  on  the  establishment  of  an  active  immunity  during  the 
period  of  incubation  of  the  disease.  In  man  the  period  of  incuba- 
tion usually  lasts  from  four  to  six  weeks,  though  it  has  been  found 
to  be  as  short  as  fifteen  days  and  as  long  as  one  year.  The  rela- 
tively long  period  of  incubation  makes  it  possible  to  establish  an 
active  immunity  before  the  symptoms  of  the  disease  develop. 

Im^munization  is  accomplished  by  repeated  injections  of  so- 
called  'Tixed  virus."  Pasteur  found  that  tissues  and  fluids  from 
rabid  animal  vary  considerabl}^  in  virulence,  and  that  the  viru- 
lence of  virus  can  be  changed  considerably.  If  successive  re-inocu- 
lations into  rabbits  be  made  from  the  virus  of  a  rabid  dog  the  virus 


82  VACCINE    AND    SERUM    THERAPY. 

is  increased  in  virulence  for  rabbits.  After  a  numljer  of  passages 
through  rabbits  the  virus  finall}^  can  no  longer  be  exalted  in  viru- 
lence. This  is  then  called  the  fixed  virus,  is  usually  obtained  from 
the  cord  and  kills  a  rabbit  in  six  to  seven  days.  This  fixed  virus 
can,  by  drying,  be  gradually  decreased  in  virulence  so  that  after 
fourteen  days  of  drying  it  has  lost  all  virulence  for  rabbits.  To 
immunize  the  individual,  virus  of  low  virulence  is  first  injected, 
after  which  injections  are  made  with  virus  of  greater  virulence  until 
at  the  end  of  the  treatment  injections  of  cord  which  has  dried  only 
from  two  to  three  days,  are  made. 

At  the  present  time  there  are  in  the  United  States  m^any  so- 
called  "Pasteur  Departments"  for  imimunizat ion  against  rabies.  The 
particular  technique  employed  in  such  departments  varies  in  some 
details,  still  the  fundamental  and  essential  principles  are  the  same. 

In  every  case  treated,  an  attempt  is  made  to  establish  definitely 
the  diagnosis  of  rabies.  Two  methods  are  employed  to  accomplish 
this :  one  is  to  examine  for  Negri  bodies  and  the  other  is  to  make 
inoculations  into  rabbits.  For  both  of  these  methods  it  is  es- 
sential that  the  head  of  the  rabid  animal  be  preserved  intact  and 
sent  to  the  laboratory  for  diagnosis.  It  is  therefore  recommended 
that  rabid  animals  be  killed  by  other  methods  than  those  which 
mutilate  the  head. 

As  soon  as  the  diagnosis  is  established,  or  if  this  is  impossible 
because  the  animal  cannot  be  found,  the  patient  should  submit  to 
treatment.  This  requires  from  two  to  four  weeks  and  consists 
usually  in  daily  injections  of  cord  of  hydrophobia  rabbits,  which 
has  been  aseptically  dried  over  caustic  potash  at  a  temperature  of 
about  23°  C.  for  varying  periods  of  time.  After  drying,  a  part  of 
the  cord  is  suspended  in  bouillon  to  which  either  0.25  per  cent  car- 
bolic acid  or  glycerine  has  been  added  as  a  preservative.  The  in- 
jections are  made  into  the  subcutaneous  tissue  and  are  usually  not 
followed  by  much  disturbance.  If  there  is  an  inflammatory  reac- 
tion, it  istobe  treated  as  any  other  cellulitis.  During  the  treatment, 
the  patient  may  go  about  his  work,  his  bowels  should  be  kept  open 
and  alcoholic  excess  avoided.  Immunization  is  not  fully  attained 
until  about  two  weeks  after  the  last  injection  of  virus. 

Immunization  is  usually  and  preferably  practiced  at  the  hos- 
pital of  Pasteur  departments.  The  New  York  City  Department  of 
Health  has  also  practised  immunization  by  sending  virus  to  physi- 


SMALL-POX    VACCIXATION.  83 

cians  outside  of  the  City  of  New  York.  In  such  cases  a  stronger 
course  of  treatment  is  followed  and  an  effort  is  made  to  limit  this 
practice  to  places  not  more  than  twenty-four  to  thirty-six  hours' 
distance  from  the  laboratory. 

The  results  of  Pasteur  treatment  or  active  ixmmunization 
against  hydrophobia  are  most  satisfactory.  Without  specific  treat- 
ment the  mortality  in  patients  bitten  by  rabid  dogs  varies  from  50 
to  80  per  cent,  while  in  patients  who  have  undergone  a  course  of 
active  immunization  the  death  rate  is  about  0.5  per  cent.  To  get 
good  results,  early  treatment  is  essential.  Bites  on  the  face  and 
head,  and  bites  by  mad  ^^olves  are  not  immunized  against  as  suc- 
cessfully as  bites  by  dogs  in  other  parts  of  the  body.  After  the 
symptoms  of  the  disease  have  developed,  active  immunization  can 
no  longer  be  expected  to  be  of  value. 

Immunization  against  rabies  has  also  been  attempted  by  the 
injection  of  anti-rabic  serum.  Babes  and  Lepp  in  1889  found  that 
they  could  protect  dogs  against  rabies  by  injection  of  serum  from 
dogs  actively  immunized  to  rabies.  Tizzoni  and  Centanni  have 
reported  favorable  results  with  anti-rabic  senmi.  Marie  has  re- 
cently advocated  the  simultaneous  injection  of  virus  and  anti-rabic 
serum.  By  this  method  it  is  possible  to  immunize  against  hydro- 
phobia more  rapidly.  The  value  of  these  newer  methods  has  not 
been  proven  so  that  at  the  present  time  conclusions  as  to  their 
value  are  impossible. 

Small-pox.  Smiall-pox  is  a  disease  for  which  the  etiological 
factor  has  not  been  determined,  although  Guanieri,  Councilman, 
Calkins  and  others  have  found  protozoan  parasites  in  the  lesions 
of  the  disease. 

For  a  long  time  it  has  been  known  that  one  attack  of  small- 
pox will  protect  against  subsequent  contraction  of  the  disease. 
This  has  led  to  the  practice  of  acquiring  immunity  against  severe 
forms  of  the  disease  by  intentional  inoculation  and  contraction  of 
mild  forms  of  the  disease. 

In  1796,  Jenner  inoculated  a  boy  with  virus  from  cow-pox  on 
a  dairy-maid's  hand,  and  found  that  in  this  w^ay  cow-pox  can  be 
transmitted  and  immunity  to  small -pox  be  produced.  Since  then 
vaccination  against  small-pox  by  inoculation  of  cow-pox  has  been 
quite  universally  adopted.  It  is  now  accepted  that  cow-pox  is  a 
modified  form  of  small-pox  and  that  small-pox  vaccination  is  a 


84  VACCINE    AND    SERl^M    THERAPY. 

process  of  active  immunization  V)y  means  of  living  organisms  at- 
tenuated by  passage  through  cows. 

The  methods  of  obtaining  virus  for  vaccination  vary.  Up  to 
1870,  virus  was  usually  obtained  from  the  pustule  of  the  vacci- 
nated individual.  Now,  however,  vaccine  is  usually  obtained  from 
young  calves  which  have  been  inoculated  on  the  abdomen  and 
thighs  with  vaccine  from  the  pustules  of  healthy  children  or  calves. 
The  pustules  on  inoculated  calves  are  cleaned  and  opened  on  about 
the  third  or  fourth  day  and  from  the  material  contained  in  them 
either  so-called  "vaccine  points"  or  "glycerinated  virus"  is  made. 
The  animals  from  which  the  vaccine  is  obtained  are  kept  as  clean 
as  possible,  asepsis  and  anti-sepsis  being  employed  as  much  as  is 
possible.  When  all  precautions  are  taken  the  vaccine  still  con- 
tains many  organisms. 

Vaccine  points  are  made  by  dipping  sterile  bone  slips  into  the 
material  from  the  pustule.  The  vaccine  is  then  dried,  kept  under 
aseptic  conditions  and  is  ready  for  use.  Glycerinated  virus  is  made 
by  softening  the  pustular  material  with  glycerine.  Glycerine  has 
the  additional  advantage  of  killing  many  bacteria  in  the  virus  and 
makes  it  possible  to  put  the  virus  up  in  tubes.  vSmall-pox  vaccine 
is  seldom  entirely  sterile,  the  organisms  present,  however,  in  a  well- 
prepared  vaccine,  consist  of  only  few  species  and  are  usually  harm- 
less. After  two  to  three  weeks  action  of  glycerine,  vaccine  is 
frequentily  free  from  bacteria. 

The  durability  of  potency  and  efficiency  of  small-pox  vaccine 
varies.  Some  vaccines  lose  their  value  in  one  month  while  others 
will  remain  efficient  for  three  to  four  months.  This  emphasizes 
the  importance  of  using  fresh  virus  for  vaccination  against  sniall- 
pox. 

The  method  of  vaccination  varies.  It  is  the  custom  now  to 
produce  scarifications  of  one-eighth  to  one-fourth  of  an  inch  in 
diamxeter  on  the  well-cleaned  arm  or  thigh.  When  vaccination  is 
made  on  the  arm,  the  area  selected  is  about  the  point  of  insertion 
of  the  deltoid  muscle.  In  cleaning  the  part  where  vaccination  is 
to  be  made  it  is  to  be  remembered  that  all  antiseptics  must  be  re- 
moved with  sterile  water  before  inoculation  is  made.  Scarifica- 
tion is  only  made  severe  enough  to  produce  an  exudation  of  serum, 
bleeding  being  avoided  as  much  as  possible.     After  scarification. 


FUTURE    OF   OPSONINS    AND    VACCINES.  85 

vaccine  on  the  point  or  from  the  tube  is  well  rubbed  in,  the  serum 
is  allowed  to  dry  and  a  dry  sterile  dressing  is  put  on. 

The  reaction  produced  by  successful  vaccination  usually  ap- 
pears after  about  three  days.  Locally,  there  is  at  first  a  papule, 
which  becomes  a  pustule  on  the  eighth  or  tenth  day.  About  the 
end  of  the  second  week  the  vesicle  changes  to  a  scab,  which  comes 
off  and  leaves  a  scar.  Constitutional  symptoms  usually  appear 
about  the  third  day  and  last  until  the  end  of  the  finst  week  after 
vaccination.  When  there  is  infection,  cellulitis  and  sloughing  fol- 
low; these  conditions  are  treated  as  any  other  infection,  but  usually 
ought  not  occur. 

The  duration  of  immunity  varies  and  is  imcertain.  The 
longest  time  that  immunity  can  be  certainly  relied  upon  is  two 
years.  The  rule  for  vaccination  which  is  generally  adopted  and 
advised  consists  in  vaccination  within  the  first  or  second  year, 
certainly  before  entering  school,  re-vaccination  within  the  tenth  to 
fifteenth  year  and  after  that  whenever  there  is  an  epidemic  of 
sniall-pox  or  possibility  of  exposure  to  the  disease. 

The  efficiency  of  vaccination  against  sriiall-pox  cannot  be 
doubted.  The  mortality  in  vaccinated  individuals  is  between  five 
and  eight  per  cent,  while  in  the  un vaccinated  thirty-five  to  forty 
per  cent  of  the  cases  terminate  fatally.  The  disease  thus  is  milder 
in  vaccinated  than  in  unvaccinated  individuals.  Moreover  fewer 
cases  of  small-pox  occur  in  the  vaccinated  than  in  those  not  vac- 
cinated against  small-pox. 


FUTURE  OF  OPSONINS  AND  VACCINES. 


As  the  method  of  determining  the  opsonic  index  stands  today 
it  is  of  questionable  value  in  diagnosis  and  treatment  of  bacterial 
diseases.  Opsonins  may  play  no  part  in  immunity;  on  the  other 
hand,  they  may  be  quite  important,  and  we  be  lacking  in  compre- 
hension of  their  value  and  their  determination.  Further  eft'orts 
should  be  made  to  study  them. 

The  treatment  of  the  bacterial  diseases  by  killed  cultures 
ought  not  be  cast  aside  because  of  the  unreliability  of  the  present 
method  of  determining  the  opsonic  index,  inasmuch  as  opsonins 
are  not  the  only  immunizing  substances  produced  by  the  injection 


86  VACCINE    AND    SERUM    THERAPY. 

of  bacterial  vaccines.  It  seems  that  the  injections  of  killed  cul- 
tures have  been  both  of  prophylactic  and  curative  value.  The 
curative  value  is  probably  limited  to  the  treatment  of  chronic 
diseases,  because  time  is  required  to  actively  immunize.  More- 
over this  method  may  be  of  especial  value  in  the  treatment  of 
infections  due  to  species  that  have  many  varieties,  for  here  the 
individual  may  be  immunized  to  the  special  organisms  causing 
the  disease.  Injections  of  killed  cultures  will  not  cure  all  infec- 
tions and  bacterial  diseases  and  in  some  cases  many  injections 
may  be  required  to  obtain  the  desired  result. 

Attention  is  again  called  to  the  fact  there  is  no  vaccine  for 
boils,  acne  or  any  other  condition,  but  there  are  vaccines  for  im- 
munization against  infections  by  the  Mic.  pyogenes  aureus,  albus 
or  citreus,  streptococci,  typhoid  bacilli,  pneumococci,  gonococci, 
etc.  Vaccine  therapy,  to  give  good  results,  requires  that  in  all 
infections  treated  the  causal  microorganisms  or  virus  must  be 
determined,  before  vaccines  are  resorted  to. 

Credit  is  to  be  given  to  Wright,  not  for  the  discovery  of  the 
method  of  immunization  by  injections  of  killed  cultures,  but  for 
its  revival.  The  employment  of  this  method  of  inducing  immunity 
and  treating  bacterial  infections  and  diseases  need  not  necessarily 
be  associated  with  opsonins  and  opsonic  immunity. 


CHAPTER  VIII. 


SERUM  THERAPY 


In  the  discussion  of  theories  of  immunity,  mention  was  made 
of  the  fact  that  in  1887  Fodor  showed  that  the  juices  of  the  nor- 
mal living  body,  especially  the  blood,  possess  bacteria-destroying 
property.  Buchner,  Behring,  and  Nuttall  were  among  the  first 
to  coroborate  the  results  of  Fodor.  It  was  later  recognized  that 
the  cell-free  blood  serum  of  normal  animals  possesses  properties 
of  destroying  bacteria.  It  is  to  be  noted,  however,  that  the  blood 
of  every  species  of  animals  will  not  destroy  all  species  of  bacteria. 
Furthermore,  while  in  certain  cases  the  degree  of  natural  immunity 
corresponds  to  the  amount  of  the  bacteria-destroying  substance 
present,  still  these  two  conditions  are  not  always  found  in  the 
same  case.  The  substance  which  destroys  bacteria,  Buchner 
called  "alexine." 

In  1888  Hericourt  and  Richet  made  the  observation  that 
blood  serum  from  an  animal  immunized  to  Staphylococcus  pyo- 
septicus,  confers  when  injected  intraperitoneally  into  rabbits, 
an  immunity  to  this  organism.  Babes  and  Lepp  in  1889  reported 
the  possibility  of  protecting  animals  against  rabies  by  the  injection 
of  body  fluid  from  animals  immunized  to  rabies.  However,  these 
results  were  given  but  little  attention  until  Behring  and  his 
pupils  systematically  investigated  the  subject. 

Behring  and  Kitasato,  in  1890,  reported  that  mice  can  be  im- 
munized to  tetanus  by  the  injection  of  blood  serum  obtained 
from  rabbits  artificially  immunized  to  tetanus.  These  investi- 
gators further  reported  that  serum  from  tetanus-immune  animals 
protects  against  the  primary  intoxication  produced  by  the  tetanus 
bacillus. 

In  1888  Roux  and  Yersin  discovered  diphtheria  toxin.  Beh- 
ring and  Wernicke,  in  1891,  showed  that  blood  serum  from  diph- 


88  VACCINE    AND    SERUM    THERAPY. 

theria  immune  guinea  pigs  or  rabbits  possesses  the  ability,  when 
injected  intraperitoneally,  to  immunize  normal  guinea  pigs  against 
diphtheria  and  a  diphtheria  infection  already  in  progress. 

In  1892  these  investigators  further  found  that  it  is  possible 
to  immunize  larger  animals  to  diphtheria,  and  demonstrated  in 
the  blood  of  these  animals  protective  and  curative  substances 
which  can  be  transferred  by  the  serum  to  other  animals.  The 
doses  necessary  to  cure  the  disease  were  found  to  be  larger  than 
those  required  to  immunize  against  the  same.  The  amount  of 
protective  and  curative  substance  produced  was  found  to  vary, 
to  some  extent  at  least,  with  the  degree  of  active  immunization 
of  the  animal  from  which  the  serum  was  obtained.  It  was  fur- 
ther found  by  Behring  that  serum  from  animals  immunized  to 
diphtheria  will  protect  against  the  toxin  of  Roux  and  Yersin. 

Based  on  these  observations,  Behring  has  formulated  a  law 
which  is  generally  knowm  as  "Behring's  Law."  According  to 
this  dictum,  the  blood  or  blood  serum  from  an  animal  possessing 
acquired  immunity  is  able  to  transmit  this  immunity  to  a  sus- 
ceptible individual  or  animal  when  this  blood  or  blood  serum  is 
injected  in  the  right  amounts  into  the  susceptible  animal  or  in- 
dividual. Individuals  naturally  immune  to  a  particular  infec- 
tious organism,  do  not  possess  in  their  blood  or  blood  serum  im- 
munizing substances  which  can  be  transmitted  to  another  in- 
dividual. Immune  substances  which  can  be  transmitted  thus 
are  not  present  naturally  but  must  be  produced  by  a  process 
of  immunization  which  consists  either  of  undergoing  the 
natural  course  of  the  disease  or  submitting  to  some  method 
of  artifical  immunization.  As  a  result  of  these  investigations, 
there  has  arisen  the  practice  of  passive  immunization,  or,  as 
it  is  more  frequently  called,  serum  therapy.  The  development 
of  methods  and  success  obtained  by  passive  immunization 
against  the  action  of  tetanus  and  diphtheria  bacilli,  led  to 
the  belief  that  it  would  be  a  relatively  easy  matter  to  make 
specific  sera  against  all  disease-producing  bacteria.  It  was 
soon  found,  however,  that  this  is  not  possible.  Acquired 
anti -toxic  immunity  has  only  a  relatively  limited  application. 
Most  pathogenic  bacteria  hold  their  special  poison  so  firmly 
within  the  cell  that  the  poison  is  only  freed  when  the  cell 
disintegrates,  or  when  certain  stimulating  conditions  as  are  found 


ANTI-TOXIC    AND   ANTI-BACTERIAL    IMMUNITY.  <S9 

in  the  lDody  exist.  Conditions  favorable  to  the  liberation  of 
toxins  have  up  to  the  present  time  been  produced  for  a  few 
bacterial  species,  so  that  most  bacteria  only  liberate  their 
toxin  in  the  tissues  of  the  body  invaded.  As  a  result  of 
immunization  to  two  widely  different  substances,  toxin  and 
bacterial  cells,  two  different  classes  of  immune  sera  have  been 
manufactured.  Of  these  two  classes,  the  anti-toxic  sera  neutralize 
certain  morbific  bacterial  products,  while  the  anti-bacterial  sera 
destroy  the  bacterial  cells.  It  is  not  to  be  inferred  from  this 
that  the  same  serum  may  not  be  both  anti -toxic  and  anti- 
bacterial, for  diphtheria  toxin  and  cells,  for  example,  may  be 
used  in  immunization.  Under  such  conditions  the  blood  serum 
obtained  will   have  both   anti-toxic   and   anti-bacterial  value. 

Acquired  anti-toxic  iinmunity  is  an  immunity  due  to  a  neu- 
tralization of  toxin  by  a  specific  anti-toxin.  The  substance  that 
is  formed  as  a  result  of  the  combination  of  toxin  and  anti-toxin 
acts  like  a  harmless  and  stable  compound.  Ehrlich,  basing  his 
observations  on  studies  of  the  union  of  toxin  and  anti-toxin, 
classes  the  anti -toxic  bodies  formed  with  the  anti-bodies  of  the 
first  order,  that  is,  the  labile  susbtance  or  complement  takes  no 
part  in  the  reaction.  In  natural  iinmunity  to  certain  toxins  the 
presence  of  specific  anti-bodies  cannot  be  demonstrated  and  un- 
der these  conditions  immunity  is  rather  a  non-susceptibility  of 
the  cells  to  the  toxin.  Metchnikoff  has  shown  that  tetanus  toxin 
circulates  freely  in  the  blood  of  naturally  immune  animals.  In 
certain  other  cases  of  natural  immunity  toxin  combines  with  cells 
that  are  of  little  importance  to  the  body  or  which  are  but  little 
affected  by  the  particular  toxin.  Acquired  anti-toxic  immunity 
thus  differs  from  natural  immunity  in  the  formation  of  immune 
bodies.  These  immune  bodies  are  found  in  the  body  fluids, 
especially  the  blood  serum  and  can  be  injected  into  other  animals 
or  individuals  and  in  this  way  confer  upon  them  a  passive 
immunity. 

Acquired  anti-bacterial  immunity  is  an  immunity  depend- 
ent upon  the  rapid  destruction  of  bacteria.  The  process 
is  one  of  solution  or  lysis.  The  anti-bodies  which  produce  lysis 
belong  to  Ehrlich's  third  order  of  immune  bodies,  that  is,  com- 
plement, which  is  easily  destroyed  by  age,  heat,  etc.,  is  necessary 
to   brincr  about  the  solution  of    bacteria.     Natural  immunity  to 


90  VACCINE    AND    SERUM   THERAPY. 

certain  bacteria  is  associated  with  the  presence  of  lytic  bodies. 
Behring  found  that  blood  serum  from  the  white  rat  which  is  nat- 
urally immune  to  the  action  of  anthrax  bacilli,  exerts  a  marked 
destructive  effect  on  anthrax  bacilli,  while  blood  from  animals 
not  immune  to  anthrax  bacilli  exert  no  such  destructive  effects 
on   these  organisms. 

On  the  other  hand  blood  serum  from  the  dog  and  the 
pigeon,  both  of  which  species  are  practically  immune  to  the 
action  of  anthrax  bacilli,  posses  no  demonstrable  bactericidal 
power.  It  is  evident  that  natural  immunity  is  not  in  all  cases 
due  to  bactericidal  action  of  blood  serum  of  non-susceptible 
animals.  Acquired  anti-bacterial  immunity  differs  from  natural 
immunity  to  the  action  of  bacteria,  in  that  definite  specific 
immune  bodies  are  present.  These  immune  bodies  are  found 
in  the  body  fluids,  especially  the  blood  serum,  and  dissolve 
bacteria. 

UNTOWARD  EFFECTS  OF  SERUM  INJECTIONS. 

In  the  conference  of  passive  immunity,  blood  and  blood  serum 
injections  are  made  almost  exclusively.  Blood  transfusions  were 
made  by  Denis  in  1667.  While  some  good  results  were  obtained, 
fever,  emboUi,  bleeding,  heamoglobinuria,  and  urticaria  were 
sometimes  produced  by  these  blood  transfusions.  Up  to  1894, 
before  diphtheria  anti-toxin  was  first  generally  used,  injections 
of  blood  and  blood  serum  were  rather  rare.  Following  the  wide- 
spread practice  of  injection  of  diphtheria  anti- toxin  in 
blood,  exanthemata  followed  in  approximately  twenty -two 
per  cent  of  the  cases.  With  the  injection  of  diphtheria  anti- 
toxin in  blood  serum,  exanthemata  were  produced  in  only  six  or 
seven  per  cent  of  the  cases.  From  .time  to  time  after  1894  cases 
have  been  reported  in  which  injections  of  diphtheria  anti-toxin  were 
followed  by  skin  manifestations.  While  the  sequellas  of  serum 
injections  are  usually  not  serious,  still  at  times  serious  symptoms 
and  even  death  have  been  reported  as  following  injections  of 
serum. 

In  1905,  V.  Pirquet  and  Schick  originated  the  term  "Serum 
Krankheit,"  or  serum  disease.  These  investigators  found  that 
serum  disease  varies,  two  rather  definite  types  of  the  disease 
being  recognized;  one  type  which  results  from  the  first  injection  of 


SERUM    DISEASE.  91 

serum  and  another  type  which  follows  the  second  and  subsequent 
injections  of  serum. 

Serum  disease  following  the  first  injection  of  serum  mani- 
fests itself  after  an  incubation  period  of  eight  to  twelve  days, 
and  is  largely  independent  of  the  amount  of  serum  injected.  Of 
the  symptoms  of  this  disease,  fever  is  the  most  constant,  lasting 
usually  during  the  entire  course  of  the  disease.  The  height  of 
fever,  however,  is  not  an  indicator  of  the  disease,  but  from  the 
curve  of  the  temperature  a  prognosis  can  be  made,  because  the 
temperature  drops  by  lysis.  Together  with  fever  there  is  usually 
an  exanthema,  appearing  most  frequently  as  an  urticaria.  This 
appears  first  about  the  point  of  injection,  later  it  is  distributed 
symmetrically  and  usually  itches  severely.  At  times  there  is  swell- 
ing of  the  glands,  especially  of  the  glands  found  in  the  region  in- 
jected. The  symptoms  of  pain  in  the  glands  are  of  prognostic 
value,  in  as  much  as  the  pain  usually  disappears  or  abates  before 
the  size  of  the  gland  decreases.  During  the  period  of  incuba- 
tion the  leucocytes  are  increased  in  number,  but  during  the  height 
of  the  disease  the  mmiber  of  leucocytes  is  markedly  diminished. 
Joint  pains  are  found  in  a  small  percentage  of  the  cases  of  serum 
disease.  The  metacarpal,  hand  and  knee  joints,  are  most  fre- 
quently effected.  Usually  these  pains  do  not  last  long.  In  cer- 
tain cases  there  is  edema,  but  albuminuria  is  seldom  or  never 
present.  The  mucous  membrane  is  seldom  effected,  which  dis- 
tinguishes the  condition  from  scarlet  fever  and  measles.  The 
disease  is  further  distinguished  from  measles  by  the  absence  of 
Koplick  spots,  coryza,  conjunctivitis  and  increased  efflores- 
cence about  the  point  of  injection.  From  scarlet  fever  the  con- 
dition is  distinguished  by  its  non-communicability  by  contact,  and 
absence  of  scaling,  nephritis  and  angina. 

The  disease  following  the  second  and  subsequent  injections 
of  serum  varies  somewhat  with  the  interval  between  injections. 
If  an  injection  of  serum  is  made  from  twelve  to  forty  days 
after  a  preceding  one,  the  incubation  period  is  very  short,  the 
disease  appearing  at  times  in  less  than  one  hour  after  injec- 
tion. If  the  interval  between  injections  is  from  forty  da^'s  to 
six  months,  there  may  be  an  immediate  reaction,  or  else  an  im- 
mediate reaction  with  another  reaction  six  to  eight  days  later. 
If  the  interval  between  injections  is  over  six  months  there  is  usu- 


92  VACCIXE    AXD    SEKU.M    THERAPY. 

ally  no  immediate  reaction,  the  symptoms  appearing  six  to  twelve 
days  after  injection.  When  the  interval  between  injections  is 
six  days  or  less,  the  disease  is  not  produced.  Serum  disease 
follows  re-injections  more  frequently  than  it  does  in  the  first  in- 
jection and  is  usually  produced  by  smaller  amounts  of  serum. 

The  "inmiediate  reaction"  occuring  when  re-injection  is  made 
within  an  interval  of  twelve  to  forty  days,  manifests  itself  in  one 
to  six  hours  after  injection  and  usually  reaches  its  maximum 
within  twenty-four  hours  after  injection.  The  "second  reaction" 
is  not  seen  after  the  first  injection  of  serum  but  occurs  most  fre- 
quently when  the  interval  between  injections  is  between  forty 
days  and  six  months.  The  incubation  period  for  the  second 
reaction  is  somewhat  shorter  than  it  is  for  serum  disease  produced 
by  first  injections.  The  symptoms  of  serum  disease  produced  by 
re-injection  are  usually  more  acute  and  general  but  of  shorter 
duration.     In  many  cases  there  is  vomiting. 

When  serum  disease  was  first  recognized  it  was  supposed  to 
be  caused  by  toxin  in  the  immune  serum.  However,  as  early  as 
1894,  Heubner  expressed  doubt  as  to  the  importance  of  specific 
immune  sera  in  the  production  of  the  disease.  Later  the  mani- 
festations of  serum  disease  were  produced  by  the  injection  of  nor- 
mal serum.  In  1906  Rosenau  and  Anderson  of  the  Hygienic 
Laboratory  of  the  Public  Health  and  Marine  Hospital  vService 
of  the  United  States,  published  a  work  in  which  experiments  were 
reported  on  "sudden  death"  of  guinea  pigs  following  serum  in- 
jections. These  investigators  found  that  re-injections  of  normal 
horse  serum  into  guinea  pigs  are  very  poisonous  if  the  interval 
between  injections  is  more  than  ten  days.  The  length  of  time  for 
which  this  h3^persusceptibility  persists  has  not  been  definitel}^ 
determined  but  lasts  at  least  as  long  as  two  years  and  two  days. 
On  the  other  hand  when  the  interval  between  injections  is  less 
than  ten  days,  normal  horse  serum  will  produce  no  such  effect. 

The  nature  and  causes  of  the  reaction  have  been  matters  of 
considerable  investigation  and  contention.  The  reaction  was  at 
first  regarded  as  one  resulting  from  the  injection  of  toxin  or  poison. 
The  long  period  of  incubation,  however,  is  explained  with  diffi- 
culty by  such  a  conception  of  the  phenomenon.  While  it  is  true 
that  to  produce  disease  by  certain  toxins,  a  period  of  incubation 
is  necessary,  this  period  is  seldom  as  long  as  eight  to  twelve  days. 


ANAPHYLOXIS.  DM 

It  can  hardly  be  conceived  that  horse  serum  has  the  abiUty  to 
increase  its  amount  of  poison  as  can  bacteria  and  other  organisms 
capable  of  self  reproduction.  Rosenau  and  Anderson  have  de- 
termined quite  definitely  that  the  reaction  is  due  to  a  difference 
in  the  susceptibility  of  individuals  and  not  the  toxicity  of  the 
serum. 

V.  Pirquet  and  Schick  conclude  that  the  phenomenon  is  due 
to  the  presence  of  specific  anti-bodies.  According  to  these  in- 
vestigators the  anti-bodies  are  not  precipitins,  for  man  does  not 
produce  the  precipitins  readily  with  horse  serum — in  children 
three  weeks  are  required  before  the  precipitins  can  be  detected 
and  are  found  in  the  blood  only  from  four  to  nine  weeks.  The 
"immediate  reaction"  which  occurs  when  the  second  injection  of 
horse  serum  is  made  twelve  to  forty  days  after  the  first,  Pirquet 
and  Schick  explain  as  being  due  to  the  already  present  serum 
anti-bodies  which  combine  with  the  immunizing  substance  in  serum 
and  produce  poisonous  substances,  which  in  turn  produce  the 
disease. 

Gay  and  Southard,  in  1907,  reported  experiments  which  in- 
dicate that  the  theory  of  Pirquet  and  Schick  is  untenable.  These 
investigators  beheve  that  "sudden  death"  in  guinea  pigs  "sensi- 
tized to  horse  serum"  is  due  to  a  substance  they  call  "anaphylac- 
tine."  This  substance  is  found  in  normal  horse  serum,  is  not 
absorbed  by  the  tissues  of  the  guinea  pig  and  is  ehminated  slowly. 
The  anaphylactine  in  the  guinea  pig  increases  the  avidity  in  the 
cells  of  the  guinea  pig,  so  that  when  more  serum  is  injected  the 
cells  are  "overwhelmed  in  the  exercise  of  their  eliminating  func- 
tions, and  functional  equilibrium  is  so  disturbed  that  local  or 
general  death  may  follow."  Rosenau  and  Anderson  had  shown 
that  the  hypersusceptibility  of  the  guinea  pig  for  a  certain  protein 
is  manifested  upon  a  second  injection  of  the  corresponding  protein. 
Later  they  showed  that  the  substance  anaphylactine  is  specific 
in  the  same  sense.  These  investigators  could  not  demonstrate 
this  substance  in  the  blood  of  the  sensitized  guinea  pig  during  the 
incubation  period,  nor  at  any  time  in  the  blood  serum  of  man, 
monkeys  and  cats. 

The  investigations  on  the  phenomenon  of  hypersusceptibility 
of  guinea  pigs  to  horse  serum  in  connection  with  serum  disease 
in  man,  has  lead  to  a  considerable  advancement  in  the  m.ethods 


94  VACCINE    AND    SERUM    THERAPY. 

of  administration  of  specific  immune  sera.  While  anaphylactine 
has  not  been  found  in  the  blood  serum  of  man,  later  investigation 
may  show  that  the  phenomenon  of  serum  disease  in  man  corresponds 
to  sudden  death  and  anaphylaxis  in  guinea  pigs. 

The  markedly  beneficial  results  of  injections  of  specific  im- 
mune sera  in  the  prevention  and  cure  of  certain  diseases,  have 
so  far  outweighed  the  undesirable  and  serious  results  which  have 
followed  serum  therapy,  that  most  physicians  have  given  little 
attention  to  the  contra-indications  of  serum  injection.  As  the 
indications  and  contra-indications  for  injection  of  immune  serum 
are  better  understood  by  physicians  fewer  objections  to  serum 
therapy,  scientifically  applied,  will  be  made.  That  serious  ob- 
jections to  the  injection  of  immune  sera  have  some  foundation, 
is  evidenced  by  the  fact  that  Rosenau  and  Anderson  have  been 
able  to  collect  a  considerable  number  of  cases  of  sudden  deaths 
in  man  after  the  injection  of  immune  serum.  It  has  been  noticed 
from  the  first  that  in  the  cases  of  sudden  death  following  the  in- 
jection of  diphtheria  anti-toxin  there  is  marked  respiratory  em- 
barrassment. Rosenau  and  Anderson  believe  the  essential  lesion 
of  serum  anaphylaxis  is  localized  in  the  respiratory  centers. 
In  collecting  statistics  on  this  subject  these  investigators  have 
found  two  cases,  "and  also  others  have  come  to  our  notice,"  in 
which  sudden  death  followed  the  injection  of  anti-toxin  into 
asthmatics.  From  these  observations  Rosenau  and  Anderson 
conclude  that  "the  knowledge  of  the  fact  that  injection  of  horse 
serum  into  asthmatics  may  be  attended  with  danger,  should  be 
considered  in  the  use  of  anti-toxins." 

While  there  are  some  cases  in  which  the  injection  of  horse 
serum  has  been  followed  by  sudden  death  due  to  a  hypersuscepti- 
bility  to  horse  serum,  most  of  the  deaths  have  undoubtedly  been 
due  to  other  causes.  Frequently  physicians  and  laymen  wrongly 
attribute  undesirable  conditions  to  the  use  of  diphtheria  anti- 
toxin and  other  immune  sera.  In  the  use  of  some  immune  sera, 
especially  diphtheria  anti-toxin,  recovery  from  the  disease  is  so 
rapid  and  early,  that  the  physician  and  patient  overestimate  the 
physical  condition  of  the  patient,  and  as  a  result  the  patient  is 
frequently  allowed  a  certain  amount  of  exercise.  This  at  times  is 
followed  by  unfavorable  symptoms.  The  condition  in  these  cases  is 
probably  due    to  the   action  of    diphtheria    toxin     rather    than 


INDICATIONS    OF    SERUM    INJECTIONS.  95 

of  diphtheria  anti-toxin.  The  heart  paralysis  which  develops 
in  some  cases  is  probably  only  observed  because  by  the  use  of 
diphtheria  anti-toxin  the  patient  remains  alive  long  enough  so 
that  the  heart  paralysis  may  be  observed.  The  action  of  diph- 
theria toxin  on  the  heart  and  other  organs  has  been  known  for 
some  time  and  in  serum  laboratories  it  is  not  an  unusual  occurence 
to  have  an  apparently  well  horse,  which  is  undergoing  iiTimuni- 
zation  to  the  diphtheria  bacillus  and  its  toxin,  die  very  suddenly 
after  exercise.  Post  diphtheritic  paralyses  have  been  observed 
long  before  the  introduction  of  the  use  of  diphtheria  anti-toxin, 
and  to  attribute  this  condition  to  anti-toxic  serum  is  to  attrib- 
ute a  symptom  to  a  remedy,  where  the  symptom  is  really  one 
found  in  the  disease  treated  by  the  remedy,  diphtheria  anti-toxin. 
In  all  cases  treated  with  immune  serum  the  patient  ought  to  be 
kept  quiet  for  some  time  after  injection  of  serum,  even  though 
he  has  apparently  recovered  from  the  disease. 

Following  the  good  results  obtained  in  the  treatment  of 
diphtheria  by  diphtheria  anti-toxin  and  the  protection  against 
the  disease  conferred  by  the  so-called  immunizing  dose  of 
diphtheria  anti -toxin,  the  serum  has  been  used  very  extensively. 
In  many  cases,  undoubtedly,  serum  has  been  used  w^ithout  proper 
indications.  Many  physicians  have  used  diphtheria  anti-toxin 
even  though  the  diagnosis  of  diphtheria  was  not  definitely  made, 
because  it  was  assumed  that  no  harm  could  come  from  the  use  of 
the  serum  while  great  benefits  might  result  if  the  condition  were 
one  of  diphtheria.  Some  physicians  have  injected  the  immuniz- 
ing dose  fully  as  indiscriminately ;  whole  families,  attending  physi- 
cian and  nurses  have  received  immunizing  doses  of  diphtheria 
anti-toxin.  The  indications  for  protective  and  curative  injec- 
tions of  immune  serum  ought  to  rest  on  a  better  foundation. 
This  is  especially  true  because  hypersusceptibility  to  serum,  or 
serum  disease,  is  more  frequent  following  re-injections  of  serum. 
It  is  evident  that  serum  ought  only  to  be  injected  into  the  indi- 
vidual when  certain  indications  are  present. 

Specific  treatment  with  immune  serum  is  only  indicated  when 
the  diagnosis,  and,  in  certain  cases,  the  etiological  factor  has  been 
determined.  It  frequently  happens  that  a  bacterialogical  diag- 
nosis cannot  be  made  immediately.  Under  such  conditions  the 
clinician  is  warranted  in  using    specific    serum   if   there    is   such 


96  VACCINE    AXD    SERUM    THERAPY. 

clinical  evidence  as  is  usually  found  in  infections  caused  by  the 
corresponding  organisms,  or  if  the  patient  has  been  exposed  to 
infection  from  a  previously  diagnosed  case  of  the  communicable 
disease. 

When  injections  of  immune  sera  are  to  be  made  frequently, 
and  over  long  periods  of  time,  as  is  the  case  with  most  anti-bac- 
terial sera,  the  injections  should  be  made  at  intervals  of  less  than 
ten  days,  because,  if  the  interval  is  longer  than  this,  serum  disease 
is  more  likely  to  develop. 

The  indications  for  injection  of  immunizing  doses  are  not 
clear  nor  definite.  Nurses  and  doctors,  however,  now  rarely  re- 
ceive immunizing  doses  of  immune  serum.  This  is  especially 
true  of  diphtheria  anti-toxin.  The  reasons  for  the  abandonment 
of  the  practice  of  immunizing  doctors  and  nurses,  are  based  on 
the  fact  that  doctors  and  nurses  are  better  able  to  avoid  infec- 
tion and  so  usually  do  not  contract  the  diseases  of  their  patients, 
and  because  the  protection  conferred  by  passive  immunization 
is  only  of  short  duration.  Doctors  and  nurses,  because  so 
frequently  exposed  to  infectious  diseases,  would  have  to  be 
injected  frequently  and  at  different  intervals  of  time  to  be 
immune  to  the  various  infections  of  their  patients.  Serum 
injections  at  intervals  of  more  than  ten  days,  however,  are  more 
likely  to  give  rise  to  serum  disease. 

The  custom  concerning  immunization  of  individuals  against 
possible  infection  because  of  exposure,  varies.  It  is  the  rule  of 
some  physicians,  upon  the  diagnosis  of  diphtheria  in  one  member 
of  the  family,  to  give  all  members  of  the  family  an  immunizing 
dose  of  diphtheria  anti-toxin.  In  some  of  the  large  children's 
hospitals  and  clinics  diphtheria  anti-toxin  is  given  to  all  patients 
on  admission,  this  being  done  to  avoid  the  outbreak  of  diph- 
theria epidemics.  Such  preventative  measures  hardly  seem 
warranted.  The  custom  of  most  physicians  now  is  to  give  im- 
munizing doses  of  some  specific  anti-sera  to  such  members  of  a 
family  or  household  as  are  most  likely  to  contract  the  disease; 
thus  for  example,  diphtheria  anti-toxin  is  given  only  to  the 
children  in  a  home  in  which  there  is  diphtheria.  On  this  basis 
administration  of  anti-sera  is  justifiable,  while  indiscriminate 
immunization  by  the  injection  of  serum  is  costly  and  may 
also  sensitize  individuals  to  serum,  which  might  later  make  the 


CONCENTRATION    OF    IMMUNE    SERA.  97 

use  of  serum  difficult  or  impossible  even  though  injection  of 
immune  serum  were  definitely  indicated.  While  it  is  true  that 
some  sera  are  ordinarily  injected  at  intervals  of  two  or  three 
weeks  without  the  production  of  serious  results,  still  it  frequently 
happens  that  such  a  degree  of  hypersensitization  to  serum  de- 
velops that  passive  immunization  must  be  given  up  entirely. 

Since  the  introduction  of  serum  therapy,  large  numbers  of 
persons  have  been  injected  with  immune  serum  and  great  bene- 
fits have  been  derived  from  the  use  of  different  sera.  The  cases 
of  serum  sickness,  especially  the  rashes,  have  been  relatively  fre- 
quent, yet  are  so  far  outweighed  by  the  beneficial  results  that 
they  must  not  prevent  the  physician  from  the  use  of  immune 
sera  when  they  are  indicated.  The  cases  of  sudden  death  fol- 
lowing injection  of  serum  fortunately  have  been  very  rare.  As 
further  knowledge  concerning  the  untoward  effects  of  the  injec- 
tion of  horse  serum  is  gained,  serum  disease  and  sudden  death 
will  become  less  frequent.  The  essential  points,  which  have  so 
far  been  developed,  indicate,  that  specific  sera  are  to  be  used  only 
after  a  definite  diagnosis  of  the  disease,  that  there  is  some  danger 
attending  the  injection  of  horse  serum  into  asthmatics,  and  that 
untoward  effects  of  serum  injections  are  more  frequent  when  the 
interval  between  injections  ranges  from  ten  days  to  several  years. 

CONCENTRATION  AND  PURIFICATION  OF  SERUM. 

Frequently  large  amounts  of  specific  immune  substances  are 
to  be  injected  in  the  treatment  of  infections.  Hypodermic  in- 
jection of  large  amounts  of  serum,  how-sver,  is  painful  and  more 
likely  to  produce  serum  disease,  especially  the  rashes.  These 
objectionable  features  of  serum  therapy  have  resulted  in  an 
attempt  to  produce  serum  of  greater  concentration  and  purity. 
It  has  been  found  that  bacteria  and  toxins  from  certain  cultures 
are  more  potent  and  cause  animals  to  produce  immtme  sera  of 
higher  protective  value.  As  a  result  virulent  cultures,  or  toxins 
produced  by  virulent  cultures,  have  been  used  in  active  immuni- 
zation of  the  animal  that  is  to  furnish  the  immune  serum. 

Attempts  have  been  made  to  obtain  an  increased  potency  by 
separating  the  immune  substance  from  the  non-immune  sub- 
stance of  specific  serum.  Dieudonne,  in  1897,  showed  that  the 
protieds  precipitated  from  diphtheria  anti-toxin  by  acetic  and 


98  VACCINE    AND    SERUM   THERAPY. 

carbonic  acid,  contain  none  of  the  anti-toxins.  In  this  same 
year  B  elf  ant  i  and  Carbone  found  that  with  the  globulins  precipi- 
tated by  magnesium  sulphate,  diphtheria  anti-toxin  is  thrown 
out.  Atkinson,  in  1901,  and  others  since  then,  have  shown  that 
during  immunization  the  serum-globulin  content  of  serum  in- 
creases. Gibson  and  Banzhaf,  however,  have  found  that  the 
increase  in  serum-globulin  is  not  necessarily  associated  with  the 
accumulation  of  anti-toxin  in  the  blood.  Early  in  1906  Gibson, 
working  in  the  Research  Laboratories  of  the  Department  of 
Health  of  New  York  City,  reported  practical  methods  for  con- 
centrating and  purifying  diphtheria  anti-toxins.  According  to 
this  method,  by  half  saturation  of  anti-toxic  serum  with  ammon- 
nium  sulphate,  the  globulins,  nucleo-proteids,  and  similar  sub- 
stances are  thrown  out.  These  contain  the  immune  bodies.  The 
precipitate  is  again  dissolved  in  a  saturated  solution  of  sodium 
chloride.  Now  only  the  anti-toxin  fraction  and  some  of  ''"he 
globulins  are  in  solution.  After  filtration,  acetic  acid  is  added 
to  the  filtrate  to  precipitate  the  anti-toxic  globulins  which  are 
then  filtered  off,  dried  with  paper,  dialyzed,  neutralized  and  again 
dialized  for  several  days.  After  dialysis  the  solution  is  made 
isotonic  by  the  addition  of  sodium  chloride.  The  sulution  is  then 
filtered  through  a  Berkefeld  filter  to  remove  any  bacteria  that 
may  be  present  and  chloroform  added  as  an  antiseptic.  This 
method  has  been  attempted  for  the  various  immune  sera,  but  up 
to  the  present  time  it  is  profitable  only  for  the  concentration  of 
diphtheria  and  tetanus  anti-toxins.  Recently  Banzhaf  has  found 
that  more  concentrated  anti-toxin  may  be  obtained  by  heating 
the  serum  to  57°  for  some  hours  before  separating  the  anti-toxic 
globulins.  By  Gibson's  method  the  concentration  is  increased 
from  three  to  five  times,  while  by  Banzhaf 's  from  eight  to  ten  times. 
By  the  use  of  concentrated  and  refined  serum  the  consti- 
tutional disturbances  and  rashes  are  somewhat  less  frequent  and 
not  as  severe.  The  principle  advantage  of  the  product  obtained 
by  these  methods,  however,  lies  in  the  fact  that  a  large  amount 
of  immune  substance  can  be  injected  in  small  amounts  of  material. 

DRIED  IMMUNE  SERUM. 

Relatively  recently,  immune  sera,  especially  anti-diphtheritic 
and  anti-tetanic  sera,^  have  been  dried  after  concentration  and 
refinement.     It  was  mentioned  earlier  that  when  serum  is  dried 


ORAL   ADMINISTRATION    OF    IMMUNE    SERA.  90 

soon  after  being  drawn,  the  immune  body  and  especially  the 
complement  are  preserved  longer.  Among  the  advantages  of 
dried  immune  sera  may  be  mentioned:  the  lack  of  deterioration, 
the  cheapness  of  manufacture;  the  convenience  of  carrying  about 
in  the  medicine  case;  and  the  possible  administration  of  immune 
substances  by  the  mouth.  Undoubtedly  these  products  will 
prove  to  be  of  considerable  value  and  are  not  to  be  lost  sight  of. 

ORAL  ADMINISTRATION  OF  IMMUNE  SERUM. 

In  1892  Ehrlich  discovered  that  ricin  anti-toxins  can  be  ab- 
sorbed by  the  intestinal  canal.  In  1893  Wernicke,  working  with 
Behring,  established  the  fact  that  anti -toxic  substances  in  the 
body  fluids  of  diphtheria-immune  animals  are  absorbed  by  the 
digestive  tract;  thus,  dogs  fed  on  the  meat  of  diphtheria-immune 
sheep  obtain  some  immunity  to  diphtheria.  The  possibility  of 
oral  administration  of  anti-toxic  sera  has  been  recently  advanced 
by  McClintock  and  King.  These  investigators  have  found  that 
the  best  results  by  oral  administration  of  diphtheria  anti-toxin 
are  obtained  by  the  following  method:  "One  half -hour  before 
administering  the  serum  the  child  is  given  one  glass  of  one  per 
cent  sodium  bicarbonate  solution.  When  the  anti-toxin  is  given 
there  is  added  one  minim  Fl.  Ext.  Opii  and  from  four  to  ten 
minims  of  saturated  solution  of  salol  in  chloroform.  When  pos- 
sible no  food  should  be  given  for  at  least  four  hours  before  ad- 
ministering the  serum."  This  method  is  adopted  to  inhibit  the 
digestion  of  the  anti-toxin  so  as  to  make  the  absorption  of  the 
unchanged  anti-toxin  possible.  Dried  diphtheria  anti-toxic  globu- 
lins were  found  to  give  satisfactory  results.  By  oral  adminis- 
tration of  diphtheria  anti-toxin  neither  serum  sickness  nor 
anaphylaxis  was  observed.  At  present  this  method  is  still  of 
questionable  value  and  requires  further  investigation. 


ANTI-TOXIC  SERA. 


It  was  stated  earlier  that  sera  containing  bodies  which  neu- 
tralized toxin  are  called  anti-toxic  sera.  The  immune  bodies 
are  receptors  of  the  first  order  of  Ehrlich  (see  p.  16),  which  have 
been  thrown  off  as  a  result  of  overproduction  of  certain  receptors. 


100  VACCINE    AND    SERUM    THERAPY. 

The  overproduction  of  these  receptors  occurs  as  a  result  of  the, 
presence  of  specific  toxins,  found  in  infections  with  specific  or- 
ganisms, or  after  experimental  injection  of  certain  toxins.  As  is  the 
case  with  other  anti-bodies,  anti-toxins  are  present  to  some  de- 
gree in  many  normal  individuals,  thus  diphtheria  anti-toxin  was 
found  by  Bolton  to  be  present  in  the  blood  serum  of  30  per  cent 
of  the  horses  examined,  and  in  50  per  cent  of  the  children  and  81 
per  cent  of  the  adults  examined  by  Wasserman.  Various  sub- 
stances have  been  used  to  produce  an  increase  of  specific  anti- 
toxins and  as  a  result  a  considerable  number  of  anti-toxins  have 
been  made.  Among  these  may  be  mentioned  the  anti-toxins 
which  combine  with  the  toxins  of  the  organisms  of  diphtheria, 
tetanus,  pyocyaneaus,  symptomatic  anthrax,  and  botulism,  with 
snake  and  spider  venoms,  with  ricin,  aprin  and  robin,  and 
with  the  glucosids  of  toad  stools  and  poison  ivy.  Of  the  anti- 
toxic sera,  the  anti-diphtheritic  and  anti-tetanic  have  been  of 
most    importance. 

In  the  treatment  of  diseases  for  which  anti-toxic  sera  have 
been  made,  it  must  always  be  remembered  that  anti-toxin  neu- 
tralizes prepared  toxin  and  combination  can  take  place  only  when 
the  receptors  of  the  toxins  and  anti-toxins  are  free.  As  soon  as 
the  cells  and  toxins  have  combined  and  the  body  cells  have  been 
injured  as  a  consec^uence,  no-  amount  of  anti-toxin  can  protect 
the  cell  from  the  action  of  the  poison  molecule.  Anti-toxic 
bodies  can  only  anchor  and  render  inert  or  harmless  such  toxins 
as  are  free  or  have  uncombined  receptors.  This  emphasizes  the 
importance  of  the  early  administration  of  anti-toxic  serum  be- 
cause at  this  time  the  poison  molecules  can  be  anchored  by  the 
anti-toxic  substances  instead  of  combining  with  the  cells  of  the 
body. 

DIPHTHERIA  ANTI-TOXIN. 

Ferran,  early  in  1890,  and  Fraenckel  and  Behring  later  in  the 
same  year,  published  methods  by  which  experimental  animals 
can  be  immunized  to  diphtheria.  Behring  and  Kitashima,  in 
1891,  published  methods  for  the  immunization  of  guinea  pigs  to 
diphtheria  toxin.  In  1892  Behring  and  Wernicke  emphasized 
the  presence  of  protective  substances  in  serum  of  diphtheria-im- 
mune animals.     Serum  therapy  as  applied  to  the  treatment  of 


DIPHTHERIA   ANTI-TOXIN.  101 

diphtheria  in  man  began  in  1891  and  1892.  The  first  diphtheria 
anti-toxin  was  put  on  the  market  in  August,  1894.  Since  then 
diphtheria  anti-toxin  has  been  used  extensively. 

The  method  for  making  diphtheria  anti-toxin  has  gone 
through  various  stages  of  development.  All  diphtheria  anti- 
toxin used  in  the  United  States  is  made  practically  after  the  same 
method,  which  is  as  follows:  Young,  vigorous  and  absolutely 
healthy  horses  are  immunized,  although  other  species  of  animals 
have  also  been  used.  During  immunization  the  animal  receives 
repeated  injections  of  diphtheria  toxin.  This  toxin  is  obtained 
by  growing  a  virulent  culture  of  B.  diphtheria  on  the  surface  of 
beef  bouillon  in  an  Ehrlenmeyer  flask.  After  one  week's  growth 
at  a  temperature  of  35°  to  36°  C,  the  culture  is  rendered  sterile 
by  adding  carbolic  acid  in  amounts  to  make  a  0 . 5  per  cent  car- 
bolic acid  solution.  After  48  hours  the  diphtheria  bacilli  have 
settled  to  the  bottom,  when  the  bouillon  is  filtered  through  a 
Berkefeld  filter.  The  germ-free  bouillon  contains  the  diphtheria 
toxin  and  ought  to  be  potent  enough  so  that  0.01  c.  c.  will  kill 
a  250  gram  guinea  pig  on  or  before  the  fourth  day  after 
subcutaneous  injection.  The  horse  or  animal  to  be  immunized 
receives  enough  of  this  toxin  in  bouillon  to  kill  five  thousand 
guinea  pigs  of  250  grams  each.  At  the  time  this  amount 
of  toxin  is  injected  the  horse  also  receives  an  injection  of  ten 
thousand  units  of  diphtheria  anti-toxin.  After  three  to  five  days, 
when  the  fever  has  subsided,  a  somewhat  larger  dose  of  toxin  and 
the  same  amount  of  anti-toxin  are  injected.  A  third  injection  is 
made  after  another  interval  of  from  three  to  five  days.  After 
this  usually  no  more  anti-toxin  is  injected  with  the  toxin,  the 
doses  of  which  are  constantly  increased  and  injected  at  intervals 
of  from  five  to  eight  days.  After  about  two  months  of  treatment, 
if  the  immunization  has  been  successful,  the  horse  will  usually 
tolerate  enough  toxin  at  one  injection  to  kill  one  hundred  thousand 
guinea  pigs  of  250  grams  each. 

At  the  end  of  six  weeks  to  two  months,  samples  of  blood  are 
drawn  and  tested  as  to  protective  value.  If  the  anti-toxic  value 
is  high,  the  horse  is  usually  bled  to  death  by  tapping  the  jugular 
vein.  The  withdrawal  of  serum  is  made  under  aseptic  conditions. 
The  bottles  containing  the  blood  are  slanted  and  after  four  to 
five  days  the  serum  is  drawn  off. 


102  VACCINE    AND    SERUM   THERAPY. 

By  means  of  Gibson's  or  Banzhaf's  method  the  serum  is  now 
concentrated  and  refined.  After  this  it  is  standardized,  i.  e.,  its 
strength  is  determined.  The  United  States  government  deter- 
mines the  unit  of  diphtheria  anti -toxin  in  the  United  States,  and 
the  Government  requires  that  all  diphtheria  anti-toxin  made  by 
manufacturers,  having  a  United  States  government  license,  must 
conform  to  this  standard.  This  emphasizes  the  importance  of 
using  only  diphtheria  anti-toxin  made  by  manufacturers  having  a 
United  States  Government  license.  Because  the  toxic  value  of 
diphtheria  toxin  changes  with  ageing  and  other  conditions,  the 
H3^gienic  Laboratory  of  the  U.  S.  Public  Health  and  Marine  Hos- 
pital Service,  issues  from  time  to  time  standard  anti-toxic  serum. 
With  this  serum  the  strength  of  the  toxin  used  in  determining 
the  anti-toxic  value  of  serum  is  gauged.  The  "immunity  unit," 
or  unit  of  anti-toxin,  is  the  amount  of  diphtheria  anti-toxic  serum 
which  will  just  neutralize  one  htmdred  times  the  smallest  amount 
of  toxin  necessary  to  kill  a  250  gram  guinea  pig  in  four  days. 
The  anti-toxic  value  of  serum  is  determined  by  mixing  one  hun- 
dred times  the  smallest  fatal  dose  of  fresh  diphtheria  toxin  with 
varying  amounts  of  the  diphtheria  anti-toxin,  allowing  the  mix- 
tures to  stand  for  fifteen  minutes  and  then  injecting  them  into 
250  gram  guinea  pigs.  The  smallest  amount  of  serum  which  will 
protect  a  250  gram  guinea  pig  for  more  than  four  days  against  one 
hundred  times  the  smallest  amount  of  diphtheria  toxin  necessary 
to  kill  a  250  gram  guinea  pig,  contains  one  unit  of  diphtheria  anti- 
toxin. 

At  the  time  of  bleeding  inmiunized  horses,  that  is  after  six 
to  eight  weeks  of  immunization,  the  serum  may  contain  from 
one  hundred  to  one  thousand  units  of  diphtheria  anti-toxin  per  c.  c. 
The  concentrated  and  refined  diphtheria  anti-toxin  contains  usually 
from  three  hundred  to  two  thousand  units  of  anti-toxin  per  c.  c. 
The  various  manufacturers  of  diphtheria  anti-toxin  now  furnish 
their  product  in  syringes  containing  a  determined  number  of 
units  of  anti-toxin.  These  packages  are  stamped  so  as  to  indicate 
the  date  after  which  they  can  be  exchanged,  free  of  charge,  for 
more  recently  tested  serum.  Because  of  the  degeneration  in 
potency  of  diphtheria  anti-toxin  in  the  fluid  condition,  it  is  desir- 
able that  no  sera  be  useti  after  the  date  when  they  are  to  be  ex- 
changed for  new  sera. 


DIPHTHERIA   ANTI-TOXIN.  103 

The  therapeutic  application  of  diphtheria  anti-toxin  has  given 
most  satisfactory  results,  and  is  now  recognized  and  regarded  as 
the  specific  for  diphtheria.  The  statistics  on  the  death  rate  from 
diphtheria  have  been  collected  at  many  different  times,  and  all 
indicate  that,  while  before  diphtheria  anti-toxin  was  used  over 
fifty  per  cent  of  the  cases  resulted  fatally,  since  the  •  application 
of  this  method  of  treatment,  the  death  rate  from  diphtheria  has 
fallen  below  twenty  per  cent,  and  according  to  some  statistics  to 
five  per  cent.  In  Boston  the  death  rate  of  diphtheria  from  1888 
to  1894  was  43.2  per  cent, while  from  1895, after  which  diphtheria 
anti-toxin  was  used  extensively,  to  1904,  inclusive,  the  death 
rate  has  fallen  to  11.84  per  cent.  Still  better  results  will  prob- 
ably be  obtained  as  this  method  of  treatment  becomes  more  uni- 
versal and  is  better  understood. 

The  method  of  treatment  of  diphtheria  with  diphtheria  anti- 
toxin varies.  However,  some  definite  principles  of  treatment 
have  been  established.  It  is  very  essential  that  all  cases  be  treated 
as  early  as  possible.  The  reasons  for  this  are  clear  when  it  is 
understood  that  as  toxin  is  liberated  by  diphtheria  bacilli,  it  tends 
to  combine  with  the  cells  of  the  body.  After  this  union  has  once 
taken  place  the  cell  is  rapidly  injured.  In  order  that  diphtheria 
anti-toxin  combine  with  these  toxins  it  is  essential  that  the  immune 
substances  be  present  at  the  time  of  the  liberation  of  the  toxin. 
Most  statistics  show  that  in  ordinary  cases  of  diptheria  the  death 
rate  is  nil  when  diphtheria  anti-toxin  is  injected  on  the  first  day 
of  the  disease.  Even  when  treated  with  diphtheria  anti-toxin  on 
the  second  day,  the  death  rate  is  below  five  per  cent;  however, 
when  specific  treatment  is  not  instituted  until  after  the  fourth 
day,  twenty  per  cent  of  the  patients  do  not  recover. 

The  dose  to  be  given  in  the  different  conditions  varies,  and 
no  hard  and  firm  rules  can  be  laid  down.  There  are,  however, 
some  quite  generally  accepted  rules  as  to  doses. 

The  ordinary  case  of  diphtheria,  treated  on  the  first  day  of 
the  disease,  receives  injections  of  from  two  to  three  thousand 
units  of  anti-toxin.  When  the  serum  is  not  given  until  the  second 
day,  usually  three  to  four  thousand  units  are  injected. 

In  the  pharyngeal  and  laryngeal  types  of  diphtheria  larger 
doses  are  usually  given — seldom  less  than  five  thousand  units. 


104  VACCIXE    AND    SERUM   THERAPY. 

When  the  disease  conies  under  ol)servation  hite  or  is  very 
severe  twenty  thousand  to  one  hundred  tliousand  units  may  be 
necessary.  Evidence  has  been  obtained  that  toxin  may  be  taken 
from  the  cells  with  which  it  has  combined  when  very  large  doses 
of  anti-toxin  are  injected.  No  (^ase  of  diphtheria  should  be  re- 
garded as  being  too  severe  or  too  far  advanced  to  be  treated  by 
diphtheria  anti-toxin,  but  in  such  cases  large  doses  should  be  given, 
as  small  doses  are  of  no  avail,  because  they  do  not  neutralize  all 
the  toxin  present.  The  age  of  the  patient,  unless  very  young, 
should  have  no  influence  on  the  amount  of  anti-toxin  injected. 

Diphtheria  anti-toxin,  as  any  other  therapeutic  agent,  should 
be  given  in  sufficient  quantities  to  accomplish  the  full  therapeutic 
action.  To  determine  this  the  effects  of  diphtheria  anti-toxin 
must  be  recognized.  The  results  of  injections  of  sufficient  amounts 
of  anti-toxin  are:  general  improvement  of  the  patient's  condi- 
tion, reduction  of  fever,  improvement  of  the  pulse,  but  most 
noticeable  of  all  is  the  shriveling  of  the  membrane,  decrease  of 
discharge,  and  less  fetid  odor  of  the  breath.  When  these  effects 
do  not  appear  from  six  to  eight  hours  after  injection,  more  diph- 
theria anti-toxin  should  be  injected.  Re-injections  usually  are 
larger  than  first  injections,  and  are  indicated  at  any  time  when 
the  patient's  condition  becomes  more  grave,  or  when  improve- 
ment does  not  come  in  six  to  eight  hours  after  injection.  At 
times  diphtheria  anti-toxin  does  not  give  satisfactory  results  in 
the  treatment  of  diphtheria.  In  most  of  these  cases,  however,  the 
treatment  is  begun  too  late  or  there  is  infection  with  other  organ- 
isms. Of  these  organisms  streptococci  are  most  important  and 
are  responsible  for  many  of  tlie  fatal  cases  of  diphtheria.  Such 
cases  are  at  times  treated  with  both  diphtheria  anti-toxin  and 
anti-streptococci  serum. 

Various  attempts  have  been  made  to  administer  diphtheria 
anti-toxin  by  the  mouth.  The  work  of  McClintock  and  King  on 
this  method  has  already  been  referred  to.  Before  oral  adminis- 
tration of  diphtheria  anti-toxin  can  be  generally  employed,  more 
experimentation  will  be  necessary. 

The  use  of  dried  diphtheria  anti-toxic  globulins,  which  are 
dissolved  in  salt  solution  before  injection,  is  relatively  recent. 
If  the  results  obtained  by  their  use  are  as  favorable  as  has  been 
reported,  it  may  be  expected  that  they  will  be  readily  taken  up 


IMMUNIZATION    AGAINST   DIPHTHERIA.  105 

uy  the  medical  profession.  Serum,  however,  has  the  advantage 
of  always  being  ready  for  use  and  not  requiring  sterilisation 
before  administration. 

In  some  of  the  severer  cases  of  nasal  diphtheria,  anti-toxin 
has  been  sprayed  on  the  membrane  with  some  beneficial  results. 
Frequently  too,  it  is  observed  that  diphtheria  bacilli  remains  viable 
in  the  nose  and  throat  after  an  apparent  recovery  from  the  dis- 
ease. Such  persons  frequently  are  the  cause  of  the  spread  of 
diphtheria.  Various  attempts  have  been  made  to  remove  the 
diphtheria  bacilli  from  the  nose  and  throat  of  these  apparently 
well  individuals.  In  addition  to  the  antiseptic  methods  em- 
ployed in  the  treatment  of  this  condition,  diphtheria  anti-toxin 
has  been  applied  locally.  Recently  it  has  been  proposed  to  ac- 
tively immunize  such  individuals  by  the  injection  of  bacterial 
vaccines  made  from  the  cultures  of  diphtheria  bacilli  isolated 
from  the  patient. 

Immunization  of  w^ell  individuals  against  possible  diphtheria 
infection  has  been  practiced  for  some  time.  It  has  been  quite 
definitely  proven  that  injection  of  a  relatively  small  number  of 
anti-toxic  units  will'  protect  from  four  to  six  weeks  against  in- 
fection w^ith  the  diphtheria  bacillus.  The  doses  generally  recom- 
mended are  from  three  hundred  to  five  hundred  units  in  small 
children  and  one  thousand  units  for  older  children  and  adults. 
While  the  custom  concerning  the  immunization  of  well  persons 
varies,  it  is  quite  generally  accepted  that  children  who  have  been 
exposed  to  diphtheria  should  receive  at  once  immunizing  doses  of 
diphtheria,  while  adults  seldom  are  treated  in  this  way. 


The  definitely  beneficial  results  which  have  been  obtained 
by  the  use  of  diphtheria  anti-toxin  in  the  treatment  of  diphtheria, 
ought  to  be  sufficient  to  convince  the  practitioner  that  diphtheria 
anti-toxin  should  be  used  in  practically  all  cases  of  diphtheria. 
Moreover  the  practioner  ought  to  know  enough  concerning  the 
symptoms  and  complications  of  diphtheria,  and  the  untoward 
effects  of  serum  injection  to  distinguish  the  conditions  dependent 
upon  the  disease  and  those  dependent  upon  the  serum  injected. 
It  is  not  unusual  for  the  practioner  to  diagnose  the  transient  serum 
rashes  as  erysiplas,   and  the  edema,  following  serum  injections, 


106  VACCINE    AND    SKRl'M    THERAPY. 

as  those  due  to  Bright's  disease.  Practioners  are  undoubtedly 
largely  responsible  for  the  misconceptions  of  laymen  concerning 
the  effects  of  diphtheria  anti-toxin  injections.  As  a  result  of  these 
misconceptions  frequently  consent  to  use  diphtheria  anti -toxin, 
when  it  is  definitely  indicated,  cannot  be  obtained,  and  yearly 
numbers  of  children  whose  lives  could  undoubtedly  have  been 
saved  are  carried  to  the  grave. 

TETANUS  ANTI-TOXIN. 

In  1890,  Behring  and  Kitasato  immunized  mice  and  rabbits 
to  tetanus  by  injecting  cultures  of  the  bacillus  of  tetanus.  These 
investigators  found  that  blood  from  rabbits  immunized  to  tetanus 
bacilli,  is  able  to  protect  mice  against  tetanus.  The  first  reliable 
anti-tetanic  serum  to  be  used  in  man,  was  put  onto  the  market 
in  1896. 

Theprocessof  producing  anti-tetanic  serum,  is  similar  to  that 
employed  to  produce  anti-diphtheria  serum — blood  is  drawn  from 
horses  that  have  received  injection  of  increasing  amounts  of  tetanus 
toxin.  The  toxin  injected  is  produced  by  growing  the  tetanus  bacillus 
on  bouillon.  After  ten  to  fifteen  days  of  incubation  under  anaerobic 
conditions,  the  bouillon  culture  is  filtered  through  a  Berkef eld  filter. 
The  germ  free  filtrate  contains  the  toxin,  which  is  present  usually  in 
large  amounts.  At  the  first  injection  into  the  horse  usually  one-half 
c.  c.  of  toxic  bouillon,  together  with  anti-tetanic  serum  are  in- 
jected. The  amount  of  toxin  is  increased  at  each  injection,  until 
finally  seven  hundred  to  eight  hundred  c.  c.  of  toxin  are  tolerated 
when  given  in  one  injection.  After  the  third  injection  the  anti- 
tetanic  serum  is  usually  omitted.  After  several  months  of  treat- 
ment, and  complete  recovery  from  the  last  injection,  the  horses 
are  bled  and  the  serum  is  collected.  The  serum  is  then  concen- 
trated and  refined  after  the  same  methods  that  are  used  for  diph- 
theria anti-toxin. 

The  standardization  of  tetanus  anti-toxin  until  very  recently 
has  been  indefinite  and  unsatisfactory.  At  the  present  time 
different  standards  exist  in  the  various  countries.  The  unit  of 
anti-toxin  for  tetanus  for  the  United  States  has  been  fixed  as  that 
amount  of  tetanus  anti-toxin  that  will  protect  a  350  gram  guinea 
pig  for  ninety-six  hours  against  one  thousand  times  the  smallest 
fatal  dose  of  tetanus  toxin.     In  order  that  the  standard  may  be 


TETANUS   ANTI-TOXIN.  107 

the  same  throughout  the  United  States  the  Hygienic  Laboratory 
of  the  United  States  Pubhc  Health  and  Marine  Hospital  Service 
sends  out  at  regular  intervals  a  stable  precipitated  tetanus  toxin 
which  is  called  the  "test  dose"  and  contains  one  hundred  times 
the  smallest  fatal  dose.  It  will  be  observed  that  a  unit  of 
tetanus  anti-toxin  contains  more  than  ten  times  as  much  neu- 
tralizing value  as  does  a  unit  of  diphtheria  anti-toxin.  The  var- 
ious anti-tetanic  serum  producers  furnish  their  product  in  suitable 
syringes,  and,  as  is  the  case  with  diphtheria  anti-toxin,  state  the 
date  after  which  it  is  desirable  that  the  serum  be  returned  if  not 
used. 

The  value  of  tetanus  ant i -toxin  and  the  methods  for  adminis- 
tration can  only  be  judged  after  study  of  the  action  of  the  tetanus 
bacillus  and  its  toxin.  Tetanus  in  man  is  usually  the  result  of 
the  subcutaneous  introduction  of  tetanus  bacilli,  some  foreign 
matter  and  usually  saprophytic  organisms.  Thus  the  disease 
follows  most  frequently  upon  puncture  and  contused  wounds 
as  the  result  of  injuries  made  by  machinery,  pistol  shots,  trauma- 
tism especially  in  stablemen,  infection  of  the  naval  during  or 
after  birth,  and  sometimes  as  a  result  of  the  injection  of  con- 
taminated vaccines  and  sera.  The  distribution  of  the  organism 
is  very  wide,  being  found  practically  everywhere  where  there  is 
animal  habitation. 

After  the  entrance  of  the  tetanus  bacillus,  usually  there  is 
little  change  produced  in  the  tissues  infected.  Suppuration  occurs 
practically  only  as  a  result  of  other  organisms.  Usually  the  or- 
ganisms remain  localized.  The  disease  and  its  symptoms  are 
entirely  dependent  on  the  absorption  of  the  toxins  produced  by 
the  tetanus  bacilli.  Tetanus  toxin  circulates  in  the  blood  and 
lymph,  but  shows  no  symptoms  until  it  unites  with  and  is  ab- 
sorbed by  the  end  organs  of  the  motor  nerves  and  the  central 
nervous  system.  To  reach  the  end  organs  the  toxin  is  carried 
through  the  axis  cylinders.  It  is  thus  seen  that  when  the  symp- 
toms of  the  disease  appear,  the  toxins  have  already  combined  with 
the  cells,  are  exerting  their  toxic  effects  on  them  and  can  no  longer 
combine  with  the  anti-toxin  which  is  not  taken  up  by  the  central 
nervous  system.  In  cases  not  treated  with  tetanus  anti-toxin.  Rose 
has  found  that  ninety-one  per  cent  of  the  cases  in  which  the  incuba- 
tion   period  is  short,  terminate  fatally;  81.3  per  cent  of  those 


108  VACCINE    AND    SERUM   THERAPY. 

having  a  medium  incubation  period  die,  while  of  the  cases  with  a 
long  incubation  period,  52.9  per  cent  of  the  patients  die. 

Tetanus  anti -toxin  can  only  bind  tetanus  toxin  before  it  is 
taken  up  by  the  nerve  cells  and  axis  cylinder.  Frequently,  how- 
ever, the  disease  is  not  suspected  until  after  the  symptoms  appear. 
Because  of  this  the  value  of  tetanus  anti-toxin  lies  mainly  in  its  pro- 
phylactic application.  As  a  preventative  of  tetanus,  tetanus  anti- 
toxin has  proven  to  be  of  great  value.  However  to  obtain  bene- 
ficial results  mjections  should  be  made  early,  as  soon  after  in- 
fection as  IS  possible,  and  inasmuch  as  immunity  lasts  only  two 
to  three  weeks,  the  injections  should  be  repeated  as  long  as  danger 
of  infection  exists.  The  immunizing  dose  which  is  generally 
used  consists  of  fifteen  hundred  immunity  units.  The  wounds 
after  w^hich  immunizing  doses  of  tetanus  anti-toxin  are  to  be 
used  are  those  made  by  bums  with  toy  pistols,  fire-works,  pistol 
shot  wounds,  injuries  occuring  while  working  about  the  stable 
or  infected  with  fertilized  garden  earth  or  manure.  The  value  of 
this  method  cannot  be  definitely  determined,  for  if  tetanus  anti- 
toxin is  used  successfully,  it  is  difficult  to  decide  whether  or  not 
there  has  been  infection  with  the  tetanus  bacillus.  It  is  the  be- 
lief of  some,  however,  that  tetanus  can  be  prevented  entirely  if 
after  all  injuries  of  the  kind  likely  to  be  infected  with  tetanus 
bacilli  immunizing  doses  of  tetanus  anti-toxin  are  injected  early. 
Jordan  states  that  in  the  United  States  in  1903,  there  were  4,449 
Fourth  of  July  injuries,  of  which  406  cases  died  of  tetanus ;  while 
in  1907,  when  tetanus  anti-toxin  was  used  quite  universally  for 
such  wounds,  there  were  only  sixty-two  deaths  caused  by  tetanus, 
following  4,413  Fourth  of  July  injuries. 

The  use  of  tetanus  anti-toxin  as  a  curative  measure  has  not 
given  universal  success.  This  is  undoubtedly  due  to  the  fact 
that  after  the  symptoms  appear  the  tetanus  toxin  has  already 
combined  with  and  injured  the  nerve  cells,  and  is  no  longer  amen- 
able to  neutralization  with  tetanus  anti -toxin.  It  is  usually 
conceded  that  if  tetanus  anti-toxin  is  not  given  within  thirty  hours 
after  the  appearance  of  symptoms,  subcutaneous  injections  of 
anti-toxins  is  of  little  avail.  When  the  disease  has  advanced, 
the  large  nerves  in  the  vicinity  of  the  infection  and  the  spinal 
canal  have  been  injected.  The  therapeutic  dose  of  tetanus  anti- 
toxin administered  varies.    If  it  is  a  case  of  short  incubation,  that 


TETANUvS   ANTI-TOXIN.  109 

is  eight  days  or  less,  injections  of  from  15,000  to  20,000  units  are 
made  every  two  to  eight  hours  until  there  is  abatement  of  the 
symptoms.  These  cases  usually  terminate  fatally,  even  when 
tetanus  anti-toxin  is  given.  The  cases  where  the  incubation  period 
is  longer  are  more  frequently  recovered  from.  In  all  cases  early 
injections  in  large  doses  are  indicated  when  the  symptoms  have 
appeared,  and  radical  methods  of  injection  are  justifiable,  because 
most  cases  without  anti-toxin  terminate  fatally.  As  a  result  of 
tetanus  anti-toxin,  the  death  rate  in  animals  as  determined  by 
Behring  has  been  found  to  be  reduced  from  88  per  cent  to  40  or 
45  per  cent.  The  curative  value  of  tetanus  anti -toxin  in  man 
cannot  be  well  demonstrated  by  statistics.  While  the  results  are 
not  nearly  as  favorable  as  those  obtained  by  treating  diphtheria 
with  diphtheria  anti-toxin,  still  tetanus  anti-toxin  ought  to  be 
used  in  all  cases  of  tetanus. 

Relatively  recently  a  dried  tetanus  anti -toxin  has  been  made. 
This  powder  is  used  principally  as  a  dressing  for  wounds  which 
are  likely  to  be  injected,  but  it  may  also  be  used  for  injection 
after  dissolving  in  salt  solution.  For  the  latter  use  it  has  the  ob- 
jection that  it  dissolves  with  difficulty.  While  relatively  little 
is  known  concerning  it,  it  has  advantages  similar  to  those  of  dried 
diphtheria  anti-toxin  and  may  prove  of  considerable  value  to  the 
medical  profession. 

As  a  result  of  the  decreased  number  of  cases  of  tetanus  fol- 
lowing Fourth  of  July  wounds,  when  immunizing  doses  of  tetanus 
anti-toxin  are  given,  the  physician  should  in  all  cases  of  such  injury 
administer  to  the  patient  an  immunizing  dose  of  tetanus  anti- 
toxin. Moreover  all  wounds  in  which  tetanus  infection  might 
occur,  should  be  thoroughly  cleansed  and  the  patient  immvmized 
with  tetanus  anti -toxin.  When  s^^mptoms  of  tetanus  have  de- 
veloped the  patient  ought  to  have  every  advantage  of  any  bene- 
fits that  may  be  derived  from  the  use  of  large  and  repeated  in- 
jections of  tetanus  anti-toxin. 


ANTI-BACTERIAL  SERA. 


Sera  containing  substances  which  result  in  the  death  and 
destruction  of  bacteria  are  called  anti-bacterial  sera.  Acquired 
anti-bacterial  immunity  depends  on  destruction  of  bacteria  be- 


110  VACCINE    AND    SERUM   THERAPY. 

fore  they  have  had  sufficient  chance  for  multiplication  and  pro- 
duction of  poisons  which  kill  the  cells  in  the  body.  For  this  reason 
anti-bacterial  sera  are  frequently  called  bactericidal  sera.  In 
the  serum  of  individuals  and  animals  that  have  acquired  anti- 
bacterial immunity,  various  kinds  of  anti-bodies  are  found.  The 
best  known  of  these  bodies  are  bacteriolysins,  opsonins,  precipi- 
tins and  agglutinins.  These  prepared  anti-bodies  are  introduced 
into  the  body  in  passive  immunization.  The  importance  and 
value  in  immunity  of  all  of  these  anti-bodies  is  not  known,  but  it 
is  generally  accepted  that  agglutinins  and  precipitins  are  of  little 
or  no  value,  while  opsonins  and  bacteriolysins  are  considered  of 
importance  in  anti -bacterial  immunity.  Bacteriolytic  immune  sera 
depend  for  their  action  on  specific  substances  called  '  'bacteriolysins, ' ' 
which  dissolve  bacteria.  The  value  of  opsonins  in  bactericidal 
sera  to  be  used  in  injections  for  the  purposes  of  conferring  passive 
immunity,  is  still  indefinitely  understood,  although  some  investi- 
gators have  attached  considerable  importance  to  their  presence 
in  sera. 

The  anti-bodies  in  anti-sera  called  agglutinins  and  precipi- 
tins belong  to  the  second  order  of  receptors,  the  bacteriolysins 
to  the  third  order,  while  in  regard  to  opsonins,  no  statement  can 
be  made  at  the  present  time — some  regarding  them  as  belonging 
to  the  second  and  others  to  the  tliird  order  of  receptors. 

Anti-sera  containing  receptors  of  either  the  second  or  third 
order  are  easily  inactivated  by  heat,  age,  acids,  etc.  After  inacti- 
vation  they  are  no  longer  able  to  produce  agglutination,  precipi- 
tation, lysis,  or  opsonification.  Tlie  loss  of  powder  to  produce  tliese 
effects  depends  on  the  destruction  of  ferment-like  substances,  which 
are  a  part  of  the  agglutinin  and  precipitin  receptors  and  are  fur- 
nished to  the  receptors  of  the  third  order  by  the  fresh  blood  serum, 
(See  page  18.)  Inactivated  bacteriolytic  serum  differs  from  in- 
activated or  aged  agglutinating  or  precipitating  serum  in  that 
on  the  addition  of  fresh  serum  the  anti-bodies  producing  lysis 
can  be  re-activated,  while  the  agglutinating  and  precipitating 
properties  cannot  be  restored  in  this  way.  Because  the  different 
specific  bacteriolytic  sera  used  in  passive  immunization  are  usually 
not  freshly  drawn  and  therefore  have  been  inactivated  by  age, 
the  individual  or  animal  immunized  must  furnish  the  complement 
or  ferment-like  substance  so  as  to  make  the  destruction  of  bacteria 


ANTI-BACTERIAL    SERA.  HI 

possible.  The  normal  individual  in  order  to  utilize  all  the  lytic 
and  possibly  the  opsonic  anti-body  in  a  specific  serum,  therefore 
must  possess  large  amounts  of  complement.  Numerous  investi- 
gators, among  which  may  be  mentioned  Ehrlich,  Morgenroth  and 
Metchnikoff,  have  found  that  in  certain  diseased  conditions  there 
is  an  actual  decrease  in  complement.  Moreover,  as  far  as  can  be 
determined,  complement  is  not  increased  during  disease  and  im- 
munization. Richardson  has  shown  that  the  serum  of  typhoid 
patients  is  not  usually  able  to  destroy  typhoid  bacilli,  but  that 
upon  addition  of  fresh  serum  from  a  normal  individual  the  typhoid 
immune  serum  is  able  to  bring  about  complete  lysis  of  typhoid 
bacilli.  Numerous  similar  observations  have  been  made  in  natural 
infections  in  man.  In  the  efficiency  of  anti-bacterial  sera  the 
presence  of  sufficient  amounts  of  complement  undoubtedly  plays 
a  large  part  and  in  certain  cases  the  lack  of  complement  probably 
is  the  cause  of  failure  of  beneficial  action  of  certain  specific  anti- 
bacterial serum.  Various  attempts  have  been  made  to  increase 
the  amount  of  complement.  Ant i -complement  has  been  used 
to  immunize  in  order  to  excite  the  over-production  of  complement. 
Injection  of  fresh  complement  with  the  immune  serum  has  been 
tried  by  some  investigators.  Efforts  have  been  made  to  preserve 
the  complement  in  immune  sera  by  freezing  the  serum  and  keeping 
it  cold  until  it  is  injected.  Recently  dried  immune  sera  have 
been  advocated  because  it  is  known  that  complement  is  preserved 
for  a  long  time  in  serum  dried  down  soon  after  it  is  drawn.  It  is 
questionable,  however,  whether  by  any  of  these  methods  enough 
complement  will  be  present  to  activate  the  amount  of  immune 
bodies  injected.  Hiss  recently  has  published  results  on  the  cura- 
tive action  of  subcutaneous  and  intraperitoneal  injection  of  ex- 
tracts of  leucocytes  from  normal  rabbits.  Of  this  leucotic  extract 
Hiss  says,  "The  action  of  the  leucocytic  extract  may  be  due  to 
the  enhancement  of  the  bacteriolytic  action  of  the  animal's  plasma 
by  the  introduction  of  complement — ^but  is  most  likely  chiefly  due 
to  the  poison  neutralizing  or  destroying  bodies." 

The  practical  application  of  specific  anti -bacterial  sera  in 
the  treatment  of  the  various  infections  in  man  has  been  tried  on 
a  large  scale.  It  has  been  found  that  by  the  early  injection  of 
serum  from  animals  actively  immunized  to  various  organisms, 
animals  can  be  rendered  non-susceptible  to  infections  with  pneu- 


112  VACCINE    AND    vSERUM    THERAPY. 

mococci,  gonococci,  streptococci,  typhoid  and  dysentery  bacilli, 
cholera  spirillae  and  many  other  microorganisms.  Based  on  these 
results,  many  different  anti-bacterial  sera  have  been  used  in  passive 
immunization  in  man.  The  results  of  ant i -bacterial  sera  in  the 
treatment  of  diseases  of  man  have,  however,  not  been  as  satis- 
factory as  those  obtained  with  specific  anti -toxic  sera.  The  im- 
portance of  complement  has  already  been  emphasized.  There  are, 
however,  other  reasons  why  anti -bacterial  sera  have  not  yielded 
as  good  results  as  anti-toxic  sera. 

Anti -bacterial  substances,  with  the  aid  of  complement,  have 
the  powder  of  destroying  bacteria  but  not  of  neutralizing  bacterial 
toxins.  For  this  reason  it  is  essential  that  anti-bacterial  sera  be 
used  early  enough  in  the  disease,  before  the  bacteria  have  had  a 
chance  to  multiply  sufficiently  and  to  set  free  enough  poison  to 
injure  and  kill  the  cells.  Early  injection  of  specific  sera  unfortu- 
nately is  not  possible  in  many  of  the  diseases  for  which  specific 
anti -bacterial  sera  have  been  used,  but  as  our  methods  of  early 
diagnosis  are  improved  earlier  administration  of  these  sera  will 
be  made.  It  is  to  be  remembered  that  it  is  of  the  utmost  impor- 
tance that  these  sera  be  vised  before  the  infecting  organisms 
have  multiplied  greatly. 

Another  reason  of  the  failure  of  anti-bacterial  sera  to  cure 
disease  is  dependent  upon  the  fact  that  as  the  bacteria  are  dis- 
solved and  destroyed  by  the  action  of  immune  substance  and  com- 
plement, the  intracellular  toxins  are  liberated.  If  the  body  cells 
are  not  able  to  cope  with  the  increased  amount  of  toxin  liberated 
upon  the  solution  of  the  bacteria,  they  will  die  and  the  gravity  of 
the  disease  be  increased.  Hiss  has  produced  evidence  to  show 
that  the  leucocytes  contain  substances  neutralizing  these  poisons. 
To  have  liberated  the  minimum  amount  of  toxin,  anti-bacteriai 
sera  should  be  administered  when  the  number  of  invading  bacteria 
is  relatively  small,  which  will  be  early  in  the  disease. 

Anti-bacterial  sera  are  standardized  with  more  difficulty  and 
less  reliably  than  are  the  anti -toxic  sera.  Attempts  have  been 
made  to  determine  the  number  of  smallest  fatal  doses  of  bacteria 
a  given  amount  of  serum  will  protect  against.  It  is  to  be  remem- 
bered, however,  that  with  living  microorganisms,  a  multiple  of 
the  fatal  dose  is  not  as  much  more  severe  than  a  single  dose  as 


ANTI-STREPTOCOCCIC    SERUM.  llo 

the  multiple  would  indicate.  Attempts  have  also  been  made 
to  determine  the  protective  value  of  specific  anti-bacterial  serum 
by  injecting  varying  amounts  of  sera,  and  bacteria,  which  have 
been  mixed  in  vitrio  and  allowed  to  act  for  some  time  before  in- 
jection. The  value  of  some  of  the  lytic  serum,  as  determined  by 
this  method,  is  high.  Frequently  it  is  not  possible  to  test  the  bac- 
tericidal power  when,  as  a  result  of  the  difficulties  of  determining 
the  amounts  of  specific  bactericidal  substances  in  the  serum,  the 
agglutinating  and  precipitating  values  are  determined.  This 
method  of  standardization  is  not  followed  because  agglutinins 
and  precipitins  are  regarded  as  bactericidal  substances  but  because 
these  anti-bodies,  to  some  extent  at  least,  accompany  bactericidal 
power. 

As  was  stated  earlier,  specific  anti-bacterial  sera  have  been 
made  by  injections  of  practically  all  the  disease  producing  bacteria. 
The  most  important  of  these  are  the  sera  which  contain  specific 
substances  by  which  they  destroy  streptococci,  meningococci, 
pneunococci,  typhoid  and  dysentary  bacilli,  and  staphlycocci. 


ANTI-STREPTOCOCCIC  SERUM. 


Anti-streptococcic  serum  was  first  made  bv  Marmorek,  in 
1895.  This  investigator  immunized  horses  by  injections  of  in- 
creasing doses  of  living  virulent  streptococci.  With  serum  from 
these  animals  he  was  able  to  confer  passive  immunity  to  rabbits 
and  also  made  attempts  to  treat  erysipelas  and  pueperal  fever  in 
the  human.  Denys  and  LeClef  have  immunized  animals  with 
bouillon  cultures  of  streptococci  and  claim  to  get  favorable  results 
upon  injection  of  serum  from  the  immunized  animals.  Van  der 
Velde,  realizing  that  there  are  different  strains  of  streptococci,  and 
that  the  anti-sera  produced  are  specific  for  the  especial  strain 
used  in  immunization,  immunized  animals  with  the  different 
strains  of  streptococcic,  thus  making  what  is  called  a  "polyvalent" 
anti-streptococcic  serum. 

The  sera  which  are  now  on  the  market  are  almost  universally 
made  by  immunizing  horses  with  repeated  injections  of  increasing 


114  VACCINE    AND    SERUM    THERAPY. 

doses  of  killed  and  later  living  bouillon  cultures  of  numerous 
strains  of  streptococci  recently  isolated  from  patients.  Several 
months  are  required  to  immunize  horses  from  which  the  immune 
serum  is  to  be  obtained.  After  testing  the  serum  for  sterility  it 
is  usually  tubed  in  suitable  syringes. 

The  method  of  action  of  anti -streptococcic  serum  is  little 
understood.  The  serum  contains  agglutinins  but  these  have  not 
been  regarded  of  value  in  immunization.  With  the  discovery  of 
bacteriotropins  by  Neufeld  and  Rimpau  and  opsonins  by  Wright 
and  Douglas  new  light  has  been  thrown  on  the  action  of  these 
sera,  for  it  has  been  found  that  in  anti -streptococcic  serum  there 
is  present  a  specific  substance  which  makes  streptococci  vulnerable 
to  ingestion  by  leucocytes. 

Standardization  and  determination  of  the  amount  of  pro- 
tective substances  in  anti-streptococcic  serum  has  not  been  suc- 
cessful, because  the  susceptibility  of  animals  to  streptococci  varies 
and  because  the  method  of  protective  action  of  the  serum  is  little 
known.  Usually  the  dose  injected  is  regulated  according  to  cubic 
centimeters  of  polyvalent  anti-streptococcic  sera.  The  potency 
of  this  serum  decreases  relatively  rapidly  so  that  it  ought  not  to 
be  used  later  than  six  months  after  the  bleeding  of  the  immunized 
horse. 

Anti-streptococcic  serum  has  been  used  especially  in  septi- 
caemia, local  infections  following  traumatisin,  streptococcic  pneu- 
monia, meningitis,  rheumatism,  erysipelas,  pueperal  sepsis,  scarlet 
fever,  secondary  infection  complicating  tuberculosis,  and  in  fact, 
in  all  streptococcus  infections. 

The  therapeutic  and  prophylactic  results  obtained  by  the  use 
of  anti-streptococcic  serum  vary  considerably  and  are  apparently 
dependent  on  various  conditions,  such  as  grade  and  kind  of  im- 
munizing serum,  day  of  disease  on  which  injections  are  made, 
amount  of  serum  injected,  virulence  of  infecting  organism,  and 
determination  of  the  invading  organism.  From  the  various  re- 
ports which  have  been  received  from  clinicians  who  have  used 
these  sera,  there  can  be  no  doubt  as  to  the  beneficial  results  which 
may  at  times  be  obtained  from  its  use.  In  order  that  good  results 
may  be  obtained,  however,  it  is  essential  that  it  be  definitely 
determined  that  the  streptococcus  is  responsible  for  the  disease^! 
condition,  that  a  high  grade  of  polyvalent  serum  be  given  early 


ANTI-MENINGOCOCCIC    SERUM.  115 

in  doses  varying  from  10  to  40  c.  c,  and  that  these  injections  be 
repeated  at  intervals  of  from  four  to  eight  hours. 

After  injection  of  anti -streptococcic  serum  the  patient  usually 
rests  more  quietly  for  several  hours.  This,  however,  need  not 
indicate  that  recovery  from  the  disease  will  follow.  The  thera- 
peutic effects  of  the  serum  are  manifested  by  the  relief  of  symp- 
toms, decline  ni  fever,  improvement  of  the  pulse,  and  subsidence 
of  the  nervous  symptoms.  These  effects  usually  appear  within 
twenty-four  hours  after  injection  if  the  serum  is  to  be  of  value, 
and  if  no  relief  comes  within  twenty-four  hours  after  two  injec- 
tions of  from  20  to  40  c.  c.  of  serum,  no  beneficial  results  from  the 
use  of  the  serum  are  to  be  hoped  for.  No  untoward  effects  of  the 
serum  are  met  with  except  the  occasional  skin  rashes  which  have 
already  been  discussed  elsewhere. 

Anti-streptococcic  serum,  even  though  its  curative  results  are 
uncertain,  ought  to  be  used  in  every  case  of  acute  streptococcus 
infection.  The  use  of  the  serum  in  scarlet  fever  is  not  universal 
but  probably  should  be  resorted  to  in  the  severer  cases.  In  the 
chronic  cases  of  discharging  sinuses,  etc.,  streptococcuc  vaccines 
are  at  times  used  to  greater  advantage  than  anti-streptococcic 
sera. 

ANTI-MENINGOCOCCIC  SERUM. 

Agglutinating  substances  were  discovered  in  1903  by  Jaeger, 
in  rabbits  experimentally  immunized  to  meningococci.  Since 
then  numerous  attempts  have  been  made  to  produce  specific 
anti-sera  to  be  used  in  passive  immunization  of  the  human.  It 
was  not  until  1906,  however,  that  injection  of  such  anti-meningo- 
coccic  serum  was  followed  with  any  degree  of  success.  In  1906 
Kolle  and  Wasserman  reported  results  on  the  use  of  specific 
meningococcus  serum  which  they  had  been  able  to  produce  in 
animals  either  by  intravenous  or  subcutaneous  injections  of 
cultures  of  meningococci  killed  by  heating  to  60°  C,  or  by  in- 
jections of  extracts  of  meningococci,  obtained  by  shaking  these 
organisms  for  four  or  five  days  in  suspension  in  distilled  water. 
Jochman  in  this  same  year  reported  results  on  a  specific  anti- 
meningococcic serum  prepared  after  similar  methods.  The  effects 
obtained  by  the  use  of  these  sera  have,   however,  not  been  as 


IIG  VACCINE    AND    SERUM    THERAPY. 

favorable  as  those  obtained  by  the  injection  of  a  serum  prepared 
by  Flexner  and  Jobling. 

The  method  of  preparation  of  the  curative  meningococcic 
serum  of  Flexner  and  Jobling,  is  to  inject  into  the  horse  first  in- 
creasing doses  of  killed  meningococci,  later  increasing  doses  of 
living  meningococci  and  finally  increasing  doses  of  an  extract  of 
meningococci.  The  extract  injected  contains  the  endotoxin  liber- 
ated by  the  action  of  a  meningococcus  autolytic  enzyme  which  as 
Flexner  has  found,  is  able  to  destroy  the  cell  substance  of  the 
meningococcus . 

The  action  of  Flexner  and  Jobling's  serum,  according  to  the 
originators,  is  three  fold.  It  exerts  an  anti-toxic  action,  is  bac- 
tericidal and  is  bacteriotropic  or  opsonifying.  Of  these  effects 
clinicians  who  have  used  the  serum  emphasize  especially  the  ap- 
parent anti -toxic  action. 

Flexner  and  Jobling  have  summarized  the  results  of  the  treat- 
ment of  400  cases  of  epidemic  cerebrospinal  meningitis.  Accord- 
ing to  this  summary  it  has  been  found  by  the  various  clinicians 
using  this  serum,  that  the  period  of  illness  can  be  shortened,  the 
chronic  lesions  largely  prevented  and  the  percentage  of  mortality 
markedly  reduced.  Probably  the  principle  reason  for  the  differ- 
ence in  efficiency  of  Flexner  and  Jobling's  serum  as  compared  to 
KoUe  and  Wasserman's  and  Jochman's  specific  sera  lies  in  the 
method  of  application.  Flexner  and  Jobling  have  emphasized  the 
importance  of  reaching  the  causal  organisms  with  the  serum.  For 
this  reason  lumbar  puncture  and  withdrawal  of  a  certain  amount 
of  cerebrospinal  fluid  is  first  made,  after  which,  without  removing 
the  needle,  injection  of  the  serum  is  made  directly  into  the  sub- 
arachnoid space  of  the  spinal  cord.  Recently  the  persistance  of 
Diplococcus  intracellularis  in  the  lateral  ventricles,  after  apparent 
death  of  the  organism  in  the  spinal  sub-arachnoid  space,  has  been 
emphasized  by  Knox  and  Sladen.  For  the  treatment  of  this  con- 
dition. Gushing  and  Sladen  have  suggested  the  advisability  of  ven- 
tricular puncture   and  injection   of  anti-meningococcus  serum. 

The  results  which  have  been  obtained  by  the  use  of  anti- 
meningococcus  serum  as  made  by  Flexner  and  Jobling,  have  been 
so  beneficial  in  acute  and  chronic  cases  of  meningitis  due  to  the 
Diplococcus  intracellularis  that  its  use  is  indicated  as  a  specific 
therapeutic  measure. 


ANTI-GONOCOCCIC    SERUM.  11 

ANTI-GONOCOCCIC  SERUM. 


Various  attempts  have  been  made  to  produce  an  anti-gono- 
coccic  serum  for  passive  immunization  of  man.  It  is  a  well  known 
fact  that  in  the  human  no  immunity  results  from  an  attack  of 
gonorrhea,  and  for  this  reason,  together  with  the  lack  of  favorable 
results  from  the  use  of  anti-gonococcic  serum,  most  investigators 
have  concluded  that  a  specific  immune  serum  for  Mic.  gonorrhoeae 
cannot  be  produced.  In  most  of  the  attempts  at  the  production 
of  anti-gonococcic  serum  old  cultures  of  the  organism  have  been 
used. 

In  1906,  Torrey  described  an  anti-gonococcic  serum  with  which 
he  had  attained  beneficial  results  in  the  treatment  of  some  cases 
of  gonorrheal  arthritis.  In  an  attempt  to  make  anti-gonococcic 
serum,  Torrey  made  the  observation  that  the  toxin  of  the  Mic. 
gonorrhoeae,  which  is  present  in  old  fluid  cultures,  is  toxic  for 
the  small  laboratory  animals.  The  toxin  for  the  gonococcus  has 
been  studied  at  various  times,  and  has  been  generally  regarded 
as  being  derived  from  the  dead  and  disintigrated  bodies  of  the 
organism,  though  by  some  it  has  been  claimed  to  be  a  true  ex- 
tracellular toxin.  Torrey  found  that  it  is  not  possible  to  im- 
munize the  small  laboratory  animals  to  this  toxin,  but  that  in 
fact  at  times  a  true  hypersusceptibility  may  develop  as  a  result  of 
injections  of  this  toxin.  This  investigator  observed,  however,  that 
animals  can  be  immunized  to  the  living  and  dead  organisms  of 
gonorrhea,  and  that  in.  the  blood  of  animals  so  immunized  agglu- 
tinins and  lysins  are  present.  Based  on  these  observations,  Tor- 
rey holds  that  the  efficiency  of  the  serum  depends  upon  specific 
bactericidal  substances  which  act  because  of  lytic  rather  than  of 
opsonifying  power.  Phagocytosis,  according  to  this  investigator, 
is  of  little  importance  in  the  destruction  of  gonococci. 

Torrey  and  Rogers  have  prepared  an  anti-gonococcic  serum 
for  use  in  treatment  of  gonorrheal  infections  in  man.  The  method 
of  preparation  of  this  serum,  as  perfected  and  recommended  by 
these  investigators,  and  used  by  the  different  serum  producers  is 
as  follows :  Strong  healthy  rams  receive  intraperitoneal  injections 
of  increasing  amounts  of  twenty-four  hour  old  ascitic  agar  cultures 
of  various,  recently  isolated,  virulent  strains  of  Mic.  gonorrhoeae. 
The  culture  is  suspended  in  salt  solution,  and  for  the  first  two 


118  VACCINE    AND    SERUM    THERAPY. 

injections,  this  suspension  is  heated  to  65°  C.  for  one-hcdf  hour 
before  the  injection  is  made.  Nine  or  ten  injections  are  usually 
necessary  to  produce  serum  of  high  value.  After  immunization 
is  completed,  the  animal  is  bled  from  the  carotid  arteries,  and  the 
serum  allowed  to  separate  out.  After  this  the  serum  is  collected, 
filtered  and  tested  for  sterility.  Immunization  is  made  in  rams 
because  blood  from  these  animals  is  apparently  little  toxic  for  the 
human.  Polyvalent  serum  is  used  because  the  imnume  bodies  for 
one  strain  are  specific  for  that  strain,  and  inasmuch  as  the  strains 
of  gonococci  vary  in  the  different  infections,  immune  bodies  for 
all  strains  must  be  present  in  the  serum. 

Standardization  of  this  serum  has  not  been  effectual.  It 
seems  quite  definite  that  this  serum  possesses  no  anti-toxic  nor 
opsonic  value,  but  is  dependent  for  its  efficiency  on  its  lytic  action. 
Because  of  a  lack  of  any  better  method  of  determining  the  immun- 
izing value  of  this  serum,  determinations  have  usually  been  made 
of  its  agglutinating  value.  The  amount  of  serum  injected  is  based 
on  cubic  centimeters  rather  than  units  of  immune  substance. 

Torrey  and  Rogers'  anti-gonococcic  serum  has  been  tried 
in  the  treatment  of  the  various  gonorrheal  infections.  From  time 
to  time  favorable  and  unfavorable  results  have  been  reported. 
The  greatest  value  of  the  serum  apparently  is  manifested  in  the 
treatment  of  the  complications  of  gonorrheal  urethritis,  such  as 
prostatitis,  epididymitis,  orchitis,  cystitis,  salpingitis,  endocarditis, 
pleuritis,  meningitis  and  especially  arthritis.  The  acute  condi- 
tions, urethritis,  vaginitis,  and  conjunctivitis,  are  little  eff'ected 
by  this  treatment. 

The  method  of  treatment  with  anti-gonococcic  serum  consists 
of  injections  of  2  c.  c.  of  serum  at  intervals  of  from  one  to  four 
days,  as  may  be  indicated  clinically.  The  injections  of  serum  are 
made  in  the  loose  subcutaneous  tissue  in  convenient  parts  of  the 
body.  In  every  case  treated  with  anti-gonococcic  serum  all  the 
other  methods  known  to  be  of  value  in  the  treatment  of  the  par- 
ticular condition  should  be  employed.  Anti-gonococcic  serum  seems 
to  cause  serum  disease  more  frequently  than  most  of  the  other 
immune  sera.  In  all  cases  it  is  to  be  remembered  that,  because 
this  method  of  treatment  frequentty  must  be  carried  over  a  long 
period  of  time,  serum  injections  must  be  made  at  intervals  of  not 
more   than  seven  to  eight  days. 


ANTI-PNEUMOCOCCIC    SERUM.  119 

While  anti-gonococcic  serum,  as  prepared  by  Torrey  and  Rogers' 
method,  has  produced  beneficial  results  in  many  conditions, 
it  is  to  be  remembered  that  improvement  and  recovery  have  not 
constantly  followed  its  injection.  Moreover  injection  of  this 
serum  ought  not  to  be  the  only  method  of  treatment  instituted. 


ANTI-PNEUMOCOCCIC  SERUM. 


Man  after  an  attack  of  pneimionia  possesses  a  certain  degree 
of  immunity.  Immunity,  however,  does  nnt  always  follow  pneu- 
monia, nor  is  it  lasting.  Early  in  the  history  of  the  diplococcus  of 
pneumonia,  attempts  were  made  to  artificially  immunize  animals, 
with  the  result  that  it  was  found  that  a  certain  degree  of  immunity 
can  be  produced.  In  as  much  as  the  pneumonococcus  does  not 
produce  an  extracellular  toxin,  anti-toxic  immunity  cannot  be 
produced  in  animals  as  a  result  of  artificial  immunization  with  this 
organism. 

The  anti-pneumococcic  serum  now  used  is  usually  taken  from 
horses  that  have  received  increasing  injections  of  dead  and  later 
of  living  cultures  of  recently  isolated  pneumococci.  Most  of  the 
sera  are  polyvalent,  the  best  known  being  those  made  by  Roemer 
.and  by  Pane.  The  method  of  action  of  anti-pneumococcic  sera 
is  little  understood.  It  is  quite  definitely  established  that  it 
possesses  no  anti-toxic  value.  The  curative  action  of  the  serum 
then,  must  depend  on  its  anti -bacterial  power.  Lysins,  until 
recent  times,  were  supposed  to  account  for  the  immunizing  value 
of  the  serum.  Neufeld  and  Rimpau  and  Wright  and  others  have 
shown  that  anti-pneumoccic  serum  contains  bacteriotropic  or 
opsonifying  substances  which  prepare  the  cocci  for  ingestion  by 
the  leucocytes. 

In  the  treatment  of  pneumonia  with  anti-pneumococcic  serum 
usually  relatively  large  amounts  of  serum  are  injected.  The 
treatment  as  recommended  by  clinicians  who  have  had  most  ex- 
perience, is  to  inject  20  c.  c.  into  the  subcutaneous  tissue  twice 
a  day  until  the  symptoms  are  relieved.  None  of  the  other  known 
methods  of  treatment  are  suspended  during  serum  treatment. 

The  results]which  have  been  attained  by  the  use  of  the  serum 
have  varied  a  great  deal.     Some  observers  have  lauded  the  use 


120  VACCINE    AND    SKRUM   THERAPY. 

of  this  serum,  but  when  all  cases  are  considered  the  results  are  not 
convincing  as  far  as  the  curative  effects  of  the  serum  are  con- 
cerned. The  reasons  for  the  failure  of  a  specific  anti-pneumococcic 
serum  are  probably  dependent  on  the  absence  of  an  anti-toxin 
in  the  serum,  injection  after  the  disease  is  far  advanced,  and  the 
difficulty  in  determining  the  species  of  organism  causing  the  pneu- 
monia. The  latter  reason  must  be  evident  to  the  clinician,  because 
the  disease  of  lobar  pneumonia  may  be  caused  by  various  differ- 
ent species  of  organisms.  Although  in  general,  the  therapeutic 
action  of  anti-pneumococcic  sera  has  not  been  marked,  still 
physicians  who  have  used  it  in  pneumonia  have  been  impressed 
with  the  general  improvement  of  the  patient,  the  lowering  of  the 
temperature  and  the  absence  of  complications,  following  its  use. 
Important  untoward  symptoms  seldom  have  developed  from  its  use. 
While  anti-pneumococcic  serum  is  not  definitely  a  curative 
measure,  the  physician  is  warranted  in  using  it  whenever  the 
patient's  condition  becomes  serious.  In  all  cases  it  is  to  be 
remembered  that  if  beneficial  results  are  to  be  obtained  wdth 
this  serum  it  must  be  injected  early. 

ANTI-TYPHOID  SERUM. 

Before  the  discovery  of  the  organism  causing  typhoid  fever, 
it  was  knowm  that  an  attack  of  typhoid  fever,  usually  gives  some 
protection  against  a  second  attack  of  the  disease.  After  the 
discovery  of  the  typhoid  bacillus  it  was  found  that  animals  repeat- 
edly injected  with  non -fatal  doses  of  this  organism  will  ultimately 
be  protected  against  otherwise  fatal  doses.  It  was  also  found 
that  by  injecting  blood  serum  from  immunized  animals,  a  passive 
immunity  against  typhoid  bacilli  can  be  conferred  to  other  animals. 
Based  on  these  results  various  anti-typhoid  sera  have  been  made 
for  the  specific  treatment  of  the  disease  of  typhoid  fever  in  man. 

Anti-typhoid  sera  are  usually  obtained  from  horses  which 
have  been  immunized  either  by  repeated  increasing  injections  of 
dead  and  living  cultures  of  typhoid  bacilli  or  typhoid  bacillus 
toxins.  Most  of  the  sera  prepared  have,  however,  not  been  anti- 
toxic but  ant i -bacterial.  Chantemesse  has  prepared  a  so-called 
anti-toxic  serum  by  injecting  horses  with  increasing  doses  of  pure 
cultures  of  typhoid  bacilli  grown  for  some  time  on  a  macerated 


ANTI-TYPHOID    SERUM.  121 

Splenic  pulp  and  defibrinated  human  blood  medium.  Tavel  by 
repeated  injections  of  two  weeks  old  bouillon  cultures,  sterilized  by 
the  addition  of  0.5  per  cent  of  carbolic  acid  has  made  a  serum 
which  is  supposed  to  possess  anti-toxic  substances.  Most  of  the 
sera  are,  however,  made  by  injecting  into  horses  and  other  animals, 
increasing  doses  of  dead  and  living  typhoid  bacilli  of  various 
strains.  For  these  sera  little  or  no  anti-toxic  value  is  claimed, 
the  immunizing  properties  being  dependent  on  the  lytic  power  of 
the  serum. 

In  the  treatment  of  typhoid  fever  witli  these  sera,  10  c.  c.  and 
frequently  larger  quantities  are  injected  daily  until  improvement 
occurs. 

The  results  following  the  use  of  anti-typhoid  serum  have  been 
disappointmg.  In  most  cases  no  effects  on  the  course  of  the 
disease  have  been  observed  followmg  the  use  of  this  serum. 
Chantemesse  in  1902,  reported,  that  by  the  use  of  his  serum  the 
death  rate  in  children  due  to  typhoid  fever  was  3  per  cent,  whereas 
the  death  rate  in  all  the  children  of  Paris  w^ho  received  no  such  serum 
injections,  was  19  per  cent.  Occasionally  observers  have  obtained 
and  reported  cases  in  which  improvement  is  rapid  after  the  in- 
jection of  anti-typhoid  serum.  As  is  the  case  with  many  of  the 
ant i -bacterial  sera,  there  is  in  general  no  influence  on  the  course 
of  the  disease.  In  some  cases,  however,  a  drop  in  the  fever  and 
improvement  in  the  pulse  and  general  conditions  have  been  re- 
ported as  resulting  from  the  use  of  this  serum.  Various  reasons 
have  been  assigned  for  the  failure  of  the  curative  action  of  the  serum, 
the  most  important  of  which  are,  the  lack  of  anti-toxic  substances 
which  can  combine  with  the  toxin  liberated  after  solution  and 
disintegration  of  the  bacilli  due  to  the  specific  lytic  substances  in 
the  serum,  and,  the  failure  of  the  body  to  supply  a  sufficient 
amount  of  complement  so  as  to  get  the  destruction  of  the  invading 
typhoid  bacilli. 

Jez  has  prepared  an  extract  from  the  spleen,  bone  marrow 
and  lymph  glands  of  animals  immunized  to  typhoid  bacilli.  This 
extract  is  usually  administered  by  the  mouth,  and  is  supposed  to 
possess  anti-toxic  properties.     The  value  of  this  extract  is  doubtful. 

Anti-typhoid  sera  are  still  in  the  experimental  stage.  With 
the  sera  now  produced  no  marked  beneficial  results  can  be  hoped 
for,  and  are  only  used  in  certain  severe  cases  of  the  disease. 


122  VACCINE    AND    SERUM    THERAPY. 

ANTI-DYSENTERIC  SERUM. 

Soon  after  the  discovery  of  the  bacillus  of  dysentery  by 
Shiga  in  1892,  the  treatment  of  bacillary  dysentery  by  the  use  of 
immune  sera  was  undertaken.  It  was  found  by  Shiga,  that  the 
organism  which  is  now  regarded  as  the  etiological  factor  in  bacillary 
dysentery  is  readily  agglutinated  with  blood  from  patients  suffer- 
ing with  this  disease.  Following  the  discovery  of  the  etiological 
importance  of  this  organism  in  the  dysenteries  in  Japan  this  or- 
ganism was  found  by  Flexner  in  the  dysenteries  in  the  Philippine 
Islands,  and  in  an  epidemic  of  dysentery  in  Germany  by  Kruse. 
In  1903,  Duval  and  Vedder  found  this  organism  in  the  discharges 
of  dysentery  patients  in  United  States,  and  in  1903,  Duval  and 
Bassett,  working  at  the  Thomas  Wilson  Sanitarium  near  Balti- 
more, Maryland,  isolated  this  organism  from  the  stools  of  children 
suffering  with  summer  diarrhoea.  Since  Shiga's  discovery  of  the 
organism,  B.  dysenteriae  has  been  found  in  association  with  dysen- 
tery in  almost  all  parts  of  the  world,  and  the  etiology  of  bacillary 
dysentery  now  seems  well  established.  While  at  first  it  was  sup- 
posed that  the  different  organisms  isolated  from  stools  in  these 
cases  were  the  same,  later  investigation  has  shown  that  there  are 
two  principal  varieties  of  the  species  of  Bacillus  dysenteriae.  Of 
these  the  original  Shiga  isolation  represents  a  strain  which  does 
not  ferment  mannite,  while  the  organism  isolated  by  Flexner 
represents  a  type  which  produces  acid  on  mannite.  The  mannite 
fermenting  type  has  now  been  found  to  include  at  least  two  and 
probably  three  different  strains.  The  original  Shiga  type  has 
been  found  especially  in  the  cases  of  epidemic  dysentery  in  adults, 
while  the  mannite  fermenting  types  are  most  frequently  found, 
althotigh  not  exclusively  or  as  the  only  type,  in  the  stools  of  chil- 
dren suffering  with  summer  diarrhoeas.  The  latter  fact  empha- 
sizes the  especial  importance  of  the  mannite  fermenting  types  of 
dysentery  bacilli  to  the  dysenteries  in  the  United  States. 

A  further  difference  has  been  found  between  the  two  main 
types;  the  mannite  fermenting  types  do  not  produce  an  extra- 
cellular toxin,  while  the  type,  not  producing  acid  on  mannite, 
produces  an  extracellular  toxin.  Because  of  these  differences 
in  the  ability  to  prodvice  extracellular  toxin,  antitoxic  immunity 
can  be  produced  by  experimental  immunization  for  the  Shiga 
type,  while  for  the  mannite  fermenting  types  the  production  of 


ANTI-DYSENTERIC    SERUM.  123 

anti-toxic  immunity  is  not  possible  experimentally.  These  differ- 
ences have  been  recognized  recently  and  only  after  much  work 
had  been  done  on  the  treatment  of  bacillary  dysentery  with  specific 
immune  sera.  Because  of  this  the  value  of  serum  therapy  in 
dysentery  can  be  determined  only  from  the  relatively  recent 
literature  on  this  subject. 

The  results  obtained  by  the  use  of  anti-dysenteric  serum  in 
the  treatment  of  dysentery  have  differed  markedly.  In  Japan, 
by  the  use  of  Shiga's  anti -dysenteric  serum,  the  mortality  of 
dysentery  has  been  reduced  from  22-26  per  cent  to  9-12  per  cent. 
Vaillard  and  Dopter  have  collected  statistics  on  two  hundred 
cases  treated  with  anti-dysenteric  serum,  in  which  cases  there  was 
a  mortality  of  only  2  per  cent.  In  the  United  States  on  the 
other  hand,  no  great  beneficial  results  have  been  attained  by  the 
use  of  the  serum  as  is  evidenced  from  the  extensive  investigations 
made  in  1903  under  the  direction  of  Dr.  Flexner. 

There  are  various  reasons  for  the  differences  in  results  which 
have  been  obtained  by  the  use  of  anti-dysenteric  serum.  The 
patients  treated  in  the  United  States  were  largely  children  under 
three  years  of  age,  while  a  large  percentage  of  the  cases  for  which 
favorable  results  are  obtained  with  serum  treatment,  occurred 
in  adults.  The  day  of  the  disease  on  which  serum  injections  are 
made  is  probably  earlier  in  such  locations  where  epidemic  dysen- 
tery is  regarded  as  a  serious  disease.  In  the  United  States  many 
cases  of  frequency  of  stool  are  only  simple  diarrhoeas,  because  of 
this  the  patient  usually  receives  no  medical  attention  until  the 
disease  is  well  advanced.  Probably  the  most  important  reason 
for  the  difference  of  the  curative  value  of  the  different  sera  is 
dependent  on  the  differences  in  the  specific  substances  in  the 
serum.  Evidently  those  sera  which  have  produced  the  most 
beneficial  results  possess  anti-toxic  properties,  while  those  used 
in  the  United  States  are  principally  or  wholly  anti -bacterial. 
Shiga  says  of  his  serum  that  it  is  "bactericidal  as  well  as  anti- 
toxic and — therefore  is  more  effective  than  anti-typhoid  serum." 
Practically  all  of  the  anti -dysenteric  sera  have  been  polyvalent, 
i .  e. ,  the  animals  furnishing  the  serum  have  been  immunized  to  the 
various  strains  and  toxins  of  dysentery  bacilli,  as  the  case  may  be. 

The  method  of  treatment  with  anti-dysenteric  serum  varies 
with  the  severity  of  the   disease.     Shiga  suggests  that   in  mild 


124  VACCINE    AND    SERUM   THERAPY. 

cases  one  dose  of  10  c.  c.  of  the  serum  be  injected.  In  cases  of 
medium  severity  two  injections  of  ten  c.  c.  each,  the  interval  be- 
tween injections  ranging  from  six  to  ten  hours,  are  recommended. 
In  the  severer  cases,  40  to  GO  c.  c.  in  all  are  to  be  injected,  but 
never  more  than  20  c.  c.  daily.  The  serum  used  in  the  United 
States  has  been  injected  in  larger  amounts,  sometimes  as  much 
as  100  c.  c.  being  injected  in  one  day. 

The  effect  of  treatment  with  Shiga's  serum  has  been  to 
decrease  the  number  of  stools,  cause  the  blood  and  pus  to  disap- 
pear from  the  stools,  restore  the  temperature  to  normal,  and  to 
lessen  the  pain  and  tenesmus.  When  this  serum  is  used  late  in 
the  disease  the  beneficial  effects  manifest  themselves  much  later. 
The  serum  used  in  the  United  States  has  not  influenced  the  course 
of  the  disease  to  any  extent,  nor  is  it  possible  to  determine  any 
particular  improvement  in  the  condition  of  patients  so  treated. 

Coyne  and  Auche  have  reported  eleven  cases  of  dysentery 
produced  by  the  Flexner,  or  mannite  fermenting,  type  of  dysentery 
bacillus,  which  were  treated  very  successfully  with  a  polyvalent 
serum.  The  curative  effects  of  this  serum  were  probably  due  to  the 
action  of  the  anti-toxin  to  the  Shiga  type  of  bacillus  dysenteriae. 

It  is  to  be  hoped  that  specific  anti-toxic  sera  can  be  made 
for  the  mannite  fermenting  group  of  dysentery  bacilli,  or  that 
Shiga  type  anti-toxin  will  be  found  to  be  efficient  in  combatting 
infections  with  the  mannite  fermenting  group  of  dysentery  bacilli . 
This  is  especially  desirable  because  the  mannite  fermenting 
organisms  are  largely  responsible  for  the  childrens'  summer 
diarrhoeas  in  the  United  States. 


ANTI -STAPHYLOCOCCIC  SERUM. 

Various  anti-staphyloccic  sera  have  been  made.  They  are 
usually  obtained  from  horses  and  other  animals  that  have  received 
repeated  injections  of  dead  and  living  cultures  of  these  organisms. 
The  method  of  its  action  is  not  clearly  understood  although  any 
protective  value  it  may  have  is  probably  due  to  lytic  and  opsoni- 
fying  power.  The  value  of  the  serum,  as  it  can  now  be  obtained, 
is  inconsiderable,  and  its  injection  in  the  treatment  of  staphy- 
lococcus infections  is  seldom  or  never  warranted. 


INDEX  OF  NAMES. 


Page 

Atkinson 98 

Babes  and  Lepp 83,  87 

Baldwin 37 

Banzhaf 98,  102,  106 

Barber 38 

Behring 100 

Behring  and  Kitasato 15,  87,  106 

Behring  and  Kitashima 100 

Behring  and  Knorr 15 

Behring  and  Wernicke 87,  100 

Belfanti  and  Carbonne 98 

Besredka 60 

Bolduan 37,  41,  47,  67 

Bolton 100 

Bordet 20 

Buchner 14,  87 

Bulloch  and  Atkin 61 

Calkins 84 

Chamberland 14 

Chantemesse 120,  121 

Chauveau 14 

Clark 54,  71 

Cole 38,  47,  48.  67 

Cole  and  Meakins 28,  76 

Covne  and  Auche 124 

D(^n 60 

Denis 90 

Deny  s 79 

Denys  and  LeClef 20,    113 

Dieudonne -'7 

Dorr 63 

Duval  and  Bassett 122 

Duval  and  Vedder 122 

Ehrlich 15,  50,  111 

Ferran 100 

Field 20 

Flexner 122,  123 

Flexner  and  Jobling 116 

Foa  and  Bonome I"* 

Fodor 14,  87 

Fraenckel 100 

Gabritschewsky 77 

Gay  and  Southard '*3 

Gibson 98,  102,  106 

Guerini 84 

Hafifkine 65 

Hamilton ^4 

Hektoen 60,  64,  67 

Herricourt  and  Richet 87 

Heubner 9- 

Hiss  and  Zinsser 45,  63,  111,  112 

Huhne  and  Neufeld r^t 

Jaeger H^ 

Jeans  and  Sellards 30,  4/ ,  48 


126  INDEX  OF  NAMES CONTINUED. 

Jenner 84 

Jez 121 

Jochman 115 

Keith (31 

Klebs 14 

Kolle  and  Wa.sserman 115 

Koch 79 

Kruse 122 

Leishman 23,  49 

Levaditi  and  Inmaii 62 

Loehleiii 60 

Madsen 50 

Maragliano 79 

Marie S3 

Marmorek •. 113 

McClintock  and  King 99,  104 

Meakins 72 

Metchnikoff 14,  20,  53,  63,  110 

Moss 38,  41,  42,  46,  47,  48,  57 

Morgenroth Ill 

Muir  and  Martin 60 

Neufeld 61,  63 

Neufeld  and  Rimpau 21,  59,  60,  1 14,  1 19 

Neufeld  and  Toepfer 61 

Noguchi 62 

Nuttall 14,  89 

Pane 119 

Park 37,  67 

Pasteur 14,  65,  82 

Petersen 45,  63 

Pfeitter 65 

Pfeiffer  and  Kolle 80 

Pirquet  and  Schick 90,  93 

Potter ; 37 

Potter,  Dittman  and  Bradley 47,  60 

Richardson Ill 

Roemer 119 

Rosenau  and  Anderson 92,  94 

Ross 34,  72,  76 

Ross  and  Johnson 77 

Roux 14 

Roux  and  Yersin 87 

Ruediger 54 

Russell 60 

Sahli 63 

Salmon  and  Smith 14 

Sauerbek 63 

Savtchenko 60 

Shiga 81,  122,  123 

Simon 37,  41,  43,  44,  45,  46,  47,  48,  60 

Simonds  and  Baldauf 62 

Solomonson 50 

Strong 46 

Tavels 121 

Tizzoni  and  Centanni .83 

Torrey  and  Rogers 117 

Trudeau 79 

Vaillard  and  Dopter 123 

V.  der  Velde 113 

AValker 31,  32,  37,  38,  39,  42,  43,  44,  45,  47 

Wasserman 100 

Wright 27,  30,  31,  35,  37,  38,  40,  44,  49,  51,  57,  59,  60,  65,  67,  72,  72,  75,  79,  119 

Wright  and  Douglas 21,  23,  27,  30,  33,  50,  59,  60,  66,  69,  70,  71,  114 

Wright  and  Ried 60 

Wright,  T.  H 29 


INDEX. 


Page 

Acne  vulgaris 7o 

Agglutinins 17,    19,    50,    53,    71,  110 

Aggressins 7,  63 

Alexines 14,  87 

Amboceptor 18,  60 

Anaphylactine 93 

Anaphylaxis 93 

Anti-bacterial  sera 106 

Anti-bodies 16,  19,  89 

Anti-dysenteric  serum 122 

historical 122 

differences  due  to  strains  of  bacilli 122 

preparation  of 123 

method  of  treatment  with 123 

results  obtained  with 124 

Anti-gonococcic  serum 117 

preparation  of 117 

method  of  treatment  with 118 

results  obtained  with 118 

Anti-meningococcic  serum 115 

Flexner  and  Jobling  serum 116 

administration  of > 116 

results  of  treatment  with 116 

Anti-pneumococcic  serum 119 

preparation  of 119 

method  of  treatment  with 119 

results  obtained  with 120 

Anti-staphylococcic  serum 124 

Anti-streptococcic  serum 113 

preparation  of 1 1  "^ 

actien  of ^ H"! 

method  of  treatment  with Hf 

results  obtained  with  and  value  of 1 1"^ 

Anti-typhoid  serum 12^ 

preparation  of 1^0 

method  of  treatment  with 120 

results  obtained  with 121 

Jez's  immune  extract ^-^ 

Anti-toxic  sera -'9 

diphtheria  anti-toxin ^^^ 

tetanus  anti-toxin ^0" 

Anti-toxic  globulins •  •  •  •  97,  102,  106 

Anti-toxin     17,   19,  53,  89,  99 

Appendix  abscesses ^J? 

Arthritis,  streptococcus i!^ 

gonococcus '  ' 

Bacillus  coli,  sensibility  of ',  , 

suspensions "^ 

vaccine '  '  '  qq   inn 

Bacillus  diphtheriae,  anti-toxni •    •  ^^'    .y 


sensibility  of. 
toxin . . . 

vaccine .        .,.-, 

Bacillus  dysenteriae  anti-serum,  sensibilitj'  of ■''^-  '-- 

vaccine. 


50 
101 
105 


SI 


128  INDEX. 

Bacillus,  pestis  sensibility  of 50 

Bacillus  pyocyaneus,  suspension  of 28 

vaccine 66 

Bacillus  tuberculosis,  immunization  to  Avith  tuberculin 78 

suspension  of i 29 

slainiufr  of 35 

vaccine  (tuberculin) 78 

Bacillus  typhosus,  anti-serum 120 

opsonic  inclex  determination  for 71 

sensibility  of 50 

vaccine 80 

Bacterial  agglutinins 17,  19,  50,  53,  71.  110 

Bactei'ial  emulsions 27,  31 

clumping  in 30,  40 

Bacterial  precipitins 17,  19,  50,  110 

Bacterial  vaccines 64 

autogenous , 68 

colon  bacillus 80 

cholera  spirillum 81 

control  of  injections  of 70,  73 

dosage  of 66 

dj'sentery  bacillus 81 

gonococcus 77 

paratyphoid  bacillus 80 

preparation  of 69 

point  of  injection  of 67 

staphylococcus 74 

streptococcus 76 

stock  culture 68 

tuberculosis  (tuberculin) 78 

typhoid  bacillus 80 

Bacteriotropins 21,  59 

Bacteriolysins 17,  19,  50,  53,  71,  89,  110,  112,  117,  119,  120,  123 

Barber's  itch 75 

Behring's  law 88 

Calculation  of  opsonic  index 35,  45 

Carbuncles 74 

Chemicals,  effect  on  opsonic  index 64 

Chicken  cholera 35 

Cholecystitis 80,  81 

Cholera,  immunization  against 65,  81 

sensibility  of  spirillum  of 50 

vaccine  against 81 

Clumping  of  bacteria  in  bacterial  suspensions 30,  44 

Complement 18,  98,  110,  112 

Complementophore  group 18 

Concentration  of  serum 97,  102,  106 

Cystitis 79,  80,  81,  118 

Cytophylic  group 8 

Diphtheria  anti-toxin 89,  100 

dried 104 

dosage 103 

oral  administration  of 104 

preparation  of 101 

standardization  of 102 

therapeutic  action  of 104 

treatment  with 103 

Diphtheria  immunization,  active 105 

with  anti-toxin 105 

Diphtheria  toxin 87,  89,  101 

Dysentery,  bacillary,  immunization  against 81 

vaccine  treatment  in 81 

serum  treatment  in 1-- 

Ehrlich's  side  chain  theory 14 

Emulsions,  bacterial 27,  31 

Endocarditis,  gonococcus 118 

streptococcus 76,  115 


INDEX.  129 

Endometritis,  colon  bacillus  infections gO 

gonococcus  infections Ug 

Erysipelas ......'....'..  54,  76 

Fixing  of  smears  for  opsonic  index  det(M-minalions 34 

Fixed  virus,  hydrophobia g2 

Furunoulosis 74    gg 

Globulins  in  immune  seru gg 

precipitation  of gg^  102,  106 

diphtheria 102 

tetanus 106 

Gonococcus  infection,  serum  therapy II7 

vaccine  therapy 77 

HaptophoFe  group 16 

Hydrophobia 65,  81 

diagnosis  of g2 

Pasteur  treatment 81,  83 

Hypersusceptibility  to  serum gO,  105 

to  vaccine 73 

Immune  sera 87 

anti-bacterial  sera 109 

anti-dysenteric  serum 122 

anti-gonococcic  serum 117 

anti-meningococcic  serum 115 

anti-pneumococcic  serum 119 

anti-staphylococcic  serum 124 

anti-streptococcic  serum 113 

anti-typhoid  serum 120 

anti-toxic  sera 99 

diphtheria    anti-toxin 100 

tetanus  anti-toxin 106 

dried 104,   109,   1 1 1 

polyvalent 113 

Immunity,  acquired 12,  65,  89 

actice 13,  65 

definition  and  classification  of 12 

passive 13,  89 

theories  of 14,  15,  20 

Immunizing  substances 13,  88,  89 

elimination  and  loss  of 20 

Infections 7 

course  of 8,  11 

elimination  of  causal  factors 11 

general  reaction  in 10 

incubation  period  in 9 

local  reaction  in 10 

Leistungskern 15 

Leucocytes,  accumulation  of 10 

in  immunity 15,  20,  21,  45,  63,  111,  112 

in  opsonic  index  determinations,  examination  of 35,  46 

source  of 26,  44 

Leucocvtosis,  relation  to  opsonic  index 62,  111,  112 

Lysis.  .' 17,  19,  50,  52,  71,  89,  110,  112,  117,  119,  120,  123 

Meningitis,  opsonic  index  in 70 

serum  treatment  of 115 

vaccine  treatment  of 70 

Micrococcus  melitensis,  sensibility  of 50 

Micrococcus  meningitidis,  anti-serum 115 

suspension  of 28 

Micrococcus  pneumoniae,  anti-serum 119 

sensibility  of 50 

suspension  of 28 

vaccine 66,  77 

Micrococcus  pyogenes,  anti-serum 124 

infection,  treatment  with  anti-serum 124 

treatment  with  vaccine 74 

suspension  of,  for  opsonic  index  determination 28 

vaccine 74 

Negative  i)hase 50,  51 


130  INDEX. 


Opsonic  index 21,  23,  24 

after  injection  of  bacterial  vaccines o7 

bacterial  eniulsions  for 27,  31,  43,  44 

calculation  of 3o,  45 

dilution  of  serum  for 41 

in  acute  otitis  media 34 

in  erysipelas 54 

in  health  and  disease 50,  53,  54 

in  tuberculosis 60,  79 

hi  typhoid  fever 54,  70 

leucocytic  emulsion  for 26,  44,  46 

mixture  of  bacteria,  leucocj^tes  and  serum  for 31,  39 

normal  serum  for 26,  39 

smears  for 33 

staining  of  smears  for 35 

sei'um  for 24,  39 

technique  for  determination  of 24,  38 

Opsonin 20,  49,  59,  110 

effect  of  chemicals  on 64 

effect  of  heat  on , 60,  61 ,  62 

effect  of  reaction  on 64 

immune  and  normal 20,  21,  59,  60,  61,  114,  119,  124 

nature  of 58 

non-bacterial 64 

specificity  of 20,  59,  61,  62 

structure  of 60,  61 

importance  in  immunity 20,  50,  53 

Oral  administration  of  anti-sera 99,  104 

Pericarditis,  gonococcus 118 

streptococcus 76,  114 

Phagocytic  index 24,  39,  45 

Phagocytosis 20,  21,  45 

causes  of 14,  20,  61 

spontaneous 30,  61 

Pneumonia 76,  119 

Pneumococcus  infections,  serum  treatment 119 

vaccine  treatment 77 

Pooled  serum 26 

Polyvalent  immune  serum 113,  123 

Positive  phase 50,  51,  71 

Precipitins 17,    19,   50,   93,    110 

Pus   10 

Rabies 81 

Reaction,  influence  on  opsonic  index 64 

Receptors 15 

first  order 16 

second  order 17 

third  order 18 

Septicaemia 9 

anti-streptococcic  serum 114 

streptococcus  vaccine 70 

Serum,  anti-bacterial 106 

anti-toxic 99 

concentration 97,   102,   106 

disease 90,  105 

dried  immune 98 

dried  diphtheria  anti-toxin 104 

dried  tetanus  anti-toxin 109 

oral  administration  of 99,  104 

pooled  normal 26 

purification  of 97,   102,   106 

therap3^ 87 

Small-pox  vaccination 84 

duration  of  immunity  from". 85 

preparation  of  vaccine  for 85 

Smears  for  determination  of  opsonic  index 33,  48 

examination  of 35,  46 

staining  of 35 


INDEX.  ];U 

yi)irilliini  eholerae,  iinmunization  against 81 

sensibility  of .^1 

Staphylococci,  suspension  for  opsonic  index  determination.  .  28 

Staphylococcus  infections,  serum  treatment  of 124 

vaccine  treatment  of 74 

Stimulins 20,  03 

Streptococci,  .suspension  for  opsonic  index  determination 2!) 

Streptococcus  infections,  serum  treatment  of 113 

vaccine  treatment  of 76 

Substance  sensibilitrice 18 

Sycosis  barbae 75 

Tetanus  antitoxin 106 

dried lOlt 

preparation  of 106 

standardization  of lOG 

treatment  with 107 

Toxin 13,  17,  87,  89,  101,  106 

produced  by  diphtheria  bacillus. 87,  101 

produced  by  tetanus  bacillus 106 

Tuberculin 78 

Tuberculosis,  treatment  with  tuberculin 79 

Typhoid  bacillus  infections,  immunization  against 65,  80 

serum  treatment  of 120 

vaccine  treatment  of 80 

Typhoid  bacillus,  sensibility  of 'A) 

suspensions  of  for  opsonic  index  determination 71 

Urethritis,  gonococcus;  serum  treatment  of 118 

vaccine  treatment  of 77 

Vaccine  therapy 65 

bacterial  vaccines 65 

Bacillus  coli 79 

Bacillus  dysenteriae 81 

Bacillus  paratyphosus 80 

Bacillus  typhosus 80 

control  of  injections 70 

dosage  of 66 

gonococcus 76 

pneumococcus 77 

preparation  of 69 

Sprillum  cholera 81 

staphjdococcus •  73 

streptococcus 75 

tuberculin 77 

of  unknown  etiology,  attenuated  virus 81 

rabies,  virus 81 

immunization  with 83 

preparation  of 82 

results  obtained  with 83 

small-pox  virus 84 

immunization  with 85 

preparation  of 84 

results  obtained  with. 85 


Diagnosis  and  Treatment  of 
Diseases  of  Women 


By 


H.  S.  Crossen,  M.  D. 


Clinical     Professor     of     Gynecology,     Medical     Department     Washington     University; 

Gynecologist    to    the    Washington    University    Hospital,    and    Chief    of    the 

Gynecological  Clinic;  Consulting  Gynecologist  to  the  Bethesda 

Hospital,     St.     Louis    Female    Hospital,    and 

St.    Louis    City    Hospital. 


816  Pages.     700  Illustrations.     Price :  Cloth  $6.00.     One-half  Morocco,  $7.50. 
Sent  anywhere  prepaid,  upon  receipt  of  Price. 


Chapter 

I. 

Chapter 

IL 

Chapter 

in. 

Chapter 

IV. 

Chapter 

V 

Chapter 

VI 

Chapter 

VII 

Chapter 

VIII 

Chapter 

IX 

Chapter 

X 

Chapter 

XI 

Chapter 

XII 

Chapter 

XIII 

Chapter 

XIV 

Chapter 

XV 

Chapter 

XVI 

Chapter 

XVII 

CONTENTS. 

Gynecologic  Examination  Methods. 

Gynecologic  Diagonsis. 

Gynecologic  Treatment. 

Diseases  of  External  Genitals  and  Vagina. 

Lacerations  and  Fistula  of  Pelvic  Floor,  Perineum,  External  Geni- 
tals and  Vagina. 

Inflammatorj'  and  Nutritive  Diseases  of  the  Uterus. 

Displacements  of  the  Uterus. 

Fibromyoma  of  the  Uterus. 

Malignant  Diseases  of  the  Uterus. 

Pelvic  Inflammation. 

Other  Affections  of  Fallopian  Tubes,  Peritoneum  and  Connective 
Tissue. 

Diseases  of  the  Ovary  and  Parovarium. 

Malformations. 

Disturbance  of  Functions. 

Invasion  of  the  Peritoneal  Cavitj'  for  the  Treatment  of  Gyneco- 
logical Diseases. 

After-Treatment  of  Operative  Cases. 

Medico-Legal  Points  in  Gjmecology. 

Appendix — Formulae — Index. 


C.  V.  MOSBY  MEDICAL  BOOK  AND  PUBLISHING  CO. 

Grand  Ave.  and  Olive  St.  ::  ::  St.  Louis,  Mo. 


(jrolden    rvules    of    Surgery 

Aphorisms  Observations  Reflections 


On  the 


Science  and  Art  of  Surgery 

A  Guide  for  Surgeons  and  Those  Who  Would  Become  Surgeons 

By 

Augustus  Charles  Bernays,  A.  M.,  M.  D. 
F.  R.  C.  S.,  England 

Late  Chief  Surgeon  Lutheran  Hospital  and  for  Twenty  Years  Professor  of  Surgery 
and  Anatomy,  St.  Louis,  Mo.,  U.  S.  A. 

230  Pages.  Handsome  Cloth  Binding.  Price,   $2.50 

Sent  Anywhere,  Prepaid,  Upon  Receipt  of  Price 


CONTENTS. 


The  Education  of  a  Surgeon. 
On  Scientific  Communications  to  the  Litera- 
ture of  Medicine  and  Surgery. 
Science  and  Surger3^ 


About  Fees. 

Off  with  the  Cloak  of  Superstition. 
Inflammation  and  the  Confusion   It  Has 
Caused. 


GOLDEN 

RULES  OF  SURGERY: 

Asepsis. 

Genito-Urinary. 

Nose. 

Anesthesia. 

Operations. 

Goitre. 

Abscesses. 

Joints. 

Shock. 

Abdomen. 

Ear. 

Oesophagus, 

Appendicitis. 

Erysipelas. 

Pelvis. 

Aneurysm. 

Gangrene. 

Rectum. 

Arterj^  Bleeding. 

Hand  and  Foot. 

Spine. 

Burns. 

Moist  Dressing. 

Throat. 

Breast. 

Mouth. 

Veins. 

Can  Minor  Surgical  Operations  Be  Done  in  OflBce? 

Death  Following  Miner  Surgical  Operations. 

Fractiures  and  Dislocations.  Therapeutic  ffints. 

Irrigation  Drainage  of  Abdominal  Cavity. 

Minor  Surgical  Cperaticns.  Stomach  and  Intestines. 


C.  V.    MOSBY    MEDICAL    BOOK   AND    PUBLISHING   CO. 

Grand  Ave.  and  OUve  Street,  St.  Louis,  Mo. 


wolden  rvules  of  r  ediat 


rics 


Aphorisms,  Observations  and  Precepts  on  the  Science  and  Art  of  Pediatrics: 
Giving  the  Practical  Rules  for  Diagnosis  and  Prognosis,  the  Essentials  of  Infant  Feed- 
ing, and  the  Principles  of  Scientific  Treatment. 

By 
JOHN  ZAHORSKY,  A.  B.,   M.  D. 

Clincal  Profos.^or  of  Pediatrics,  ^\'a^hinKt(Jll  I'nivcrsity  Medical  Department,  St.  Louis; 
Ex- President  Bethesda  Societj^;  Attending  Phy.sician  to  the  Bethe.sda  Found- 
lings' Home;  Member  of  the  American  Medical  Association  and  of  the 
St.  Louis  Academy  of  Science;  Editor  of  the  St.  Louis  Courier 
of    Medicine;    Author   of    "Baby    Incubators,"   etc. 

WITH  AN  INTRODUCTION 

By 

E.  W.   SAUNDERS,   M.  D. 

Professor  of  Diseases  of  Children  and  Clinical  Midwiferv.  Washington  L'niver.sity, 

St.  Louis,  Mo. 

370  Pages,  Silk  Cloth  Binding,  Price  $3,  sent  Post  Paid  anywhere  en  receipt  of  price. 

CONTENTS. 

Ascites.     The  Adenoid  Face. 
Acute  Pneumonic  Consolidation. 
Intestinal  Obstruction. 
Nervous  State.     Scrofula. 
Tuberculosis. 


Introduction. 
Part  I. 
General  Rules  of  Diagnosis. 
General  Rules. 
Loss  in  "\^'eight.     Appetite. 
Convulsions. 
Physical  Examination. 
Head  and  Neck. 
Some  Deformities. 
Teeth  and  Gums.      Dentition. 
The  Enanthemata. 
Vomiting.      Hematemesis. 
Diarrhea. 

Distended  Abdomen. 
Abdominal  Pain. 
Abdominal  Swellings. 
The  Nose  and  Nasopharynx. 
The  Larynx. 

Anomalies  of  Breathing.      Cough. 
The  Lungs. 

The  Heart  and  Circulation. 
The  Urine.     The  Eruptions. 
The  Nervous  System. 
Paralysis. 

Tremor,     Choreiform     Movements.      Head- 
ache, etc. 
Changes  Abo;it  the  Eyes. 
Changes  About  the  Ear. 
Clinical  Syndromes. 
FeA'er.      Chronic  Fever. 
Status  Gastricus. 
The  Typhoid  State. 
Infantile  Atrophy. 
Gastroenteric  Infection,  Diarrhea. 
Chronic  Indigestion  in  Older  Children. 
Chronic  Constipation. 
Peritoneal  Irritation. 
Severe  Anemia.     Edena. 


Impending  Heart  Failure. 

The  Syndrome  of  Cerebral  Irritations. 

Golden  Rules  cf  Prognosis. 

Part  II. 

Golden    Rules    cf    Hygiene    and    Infant 

Feeding. 

The  Nursing  Mother. 
The  Wet  Nurse. 
Artificial  Feeding. 
Feeding  the  Sick. 

Golden  Rules  cf  Treatment. 
General  Therapeutics. 
The  Newly  Born. 
Diseases  of  the  Mouth. 
The  Neck  and  Scalp. 
The  Throat. 

The  Respiratory  Organs. 
G-astroenteric  Diseases. 
Rickets  and  Scurvy. 
Lleart  and  Circulation. 
The  Blood. 

The  Genito-L'rinary  Organs. 
The  Nervous  Sj^steni. 
Specific  Infectious  Diseases. 
Malaria.      Cerebro-Spinal  Fever. 
Diphtheiia.     Intubation. 
Tuberculosis.      Pertussis. 
Mumps.      Sei:ticemia. 
Rheumatism  and  Endocarditis. 
Syphilis.      The  Exanthemata. 
The  Severe  Infectious  Fevers. 
The  Skin. 
Formularv. 


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GRAND  AVENUE  AND  OLIVE  STREET,  SAINT  LOUIS,   MISSOURI. 


Golden  Rules  of  Dietetics 


By  A.  L.  BENEDICT,  A.   M.,  M.  D. 

Consultant  in    Digestive    Diseases,  City  and    Riverside    Hospitals    and    Attendant  in 

same;  Mercy  Hospital,  Buffalo;  Member  of  the  Academy  of  Medicine  and 

of    American    Gastro-Entrological    Association,    etc.; 

Author  of    Practical  Dietetics. 

CONTENTS. 

Part  I. 

Phy.siologic  Chemistry. 

Daily  Recjuirements  of  the  Human  Body. 

Standard  Diet  in  Health. 

Quantative  Estimate  of  Diet. 

Approximate  Methods  of  Checking  Diet  Weight  and  the  Excretions. 

Transmutability  and  Reservation  of  Food. 

Waste  of  Food. 

Predigestion  of  Food. 

Emergency  Methods  of  Introducing  Nourishment. 

Preserved  Foods. 

Methods  of  Cooking. 

Compositions  of  Natural  and  Commercial  Foodstuffs. 

Food  Adjuncts. 

Purine  Bodies. 

Important  Constituents  of  Foodstuffs. 

Distinctly  Deleterious  Foodstuffs. 

General  Hygiene  of  Eating. 

Diet  Tests. 

Condensation  of  Atwatcr  &  Bryant's  Analysis  of  Foodstuffs. 

Part  II. 

Principles  of  Dietetics  According  to  General  Pathologic  Conditions. 

Infant  Feeding. 

Diet  in  Critical  Physiologic  Periods. 

Diabetis,  Glycosuria. 

Obesity  and  Leanness. 

Chronic  Diseases  of  Nitrogeneous  Metabolism. 

Diseases  of  the  Urinary  Organs. 

Diseases  of  the  Ductless  Glands. 

Diseases  of  the  Liver. 

Diseases  of  the  Pancreas. 

Diseases  of  the  Digestive  Organs. 

General  Preversion  of  Digestive  Functions. 

Functional  Intestinal  Diseases. 

Organic  Intestinal  Diseases. 

Diseases  of  the    Heart  and  Blood  Vessels. 

Blood  Diseases. 

Hemorrhagic  Diseases. 

Bone  Diseases. 

General  Principles  of  Feeding  in  Fevers. 

Infectious  and  Parasite  Diseases. 

Respiratory  Diseases. 

Skin  Diseases. 

Diseases  of  the  Nervous  System. 

Surgical  Emergencies  and  Operations. 

450  Pages,  Octavo.     Price  $3.00 


Chapter 

I. 

Chapter 

II. 

Chapter 

III. 

Chapter 

IV. 

Chapter 

V. 

Chapter 

VI. 

Chapter 

VII. 

Chapter 

VIII. 

Chapter 

IX. 

Chapter 

X. 

Chapter 

XI. 

Chapter 

XII. 

Chapter 

XIII. 

Chapter 

XIV. 

Chapter 

XV. 

Chapter 

XVI. 

Chapter 

XVII. 

Chapter 

XVIII. 

Chapter 

XIX. 

Chapter 

I. 

Chapter 

II. 

Chapter 

III. 

Chapter 

IV. 

Chapter 

V. 

Chapter 

VI. 

Chapter 

VII. 

Chapter 

VIII. 

Chajiter 

IX 

Chapter 

X. 

Chapter 

XI. 

Chapter 

XII. 

Chapter 

XIII 

Chapter 

XIV 

Chapter 

XV. 

Chapter 

XVI. 

Chapter 

XVII. 

Chapter 

XVIII. 

Chapter 

XIX. 

Chapter 

XX. 

Chapter 

XXI 

Chapter 

XXII 

Chapter 

XXIII 

Chapter 

XXIV 

C.  V.  MOSBY  MEDICAL  BOCK  AND  PUBLISHING  CO. 

Grand  Ave.  and  Olive  Street,  St.  Louis,  Mo. 


A  VERY 
YOUNG    OVUM    IN   SITU 

By 

G.    Leopold, 

Dresden 
Authorized  Translation  By 

W.  H.  VOGT,  A.  M.,  M.  D. 

Obstetrician  and  Gynecologist,  Lutheran  Hospital,  St.  Louis,  Mo. 


65  Pages  Text.        35  Pages  Lithographic  Illustrations  in  Colors 

Price,  Cloth  $3.50 


Publisher's  Announcement 

The  importance  of  an  understanding  of  embryolog}'  is  becoming  more  and  more 
apparent.  The  student  now  realizes  that  anatomy  is  much  better  understood — where 
it  is  worked  out  from  the  standpoint  of  embryonic  development — than  where  it  is 
learned  in  its  crude  state  in  the  dissecting-room.  The  surgeon  realizes  that  he  can  bet- 
ter grasp  the  relationship  of  structures  when  he  is  familiar  ^^^th  their  formation  from 
the  embrj'o.  The  scheme  of  development  as  Avorked  out  by  Leopold  represents  the  latest 
work  along  this  line.  The  work  is  most  scientific  and  cannot  fail  to  interest  all  who 
are  seeking  the  fundamental  truths  of  embryonic  development. 


THE   C.   V.   MOSBY  MEDICAL 
BOOK  AND  PUBLISHING  CO. 

Grand  Avenue  and  Olive  Sreet,  ::  St.  Louis,  Missouri. 


rLxamination    of     1  he    lL 


ar 


By 
Selden  Spencer,  A.  B.,  M.  D. 

Instructor  of  Otology  in  the  Washington  University  Medical  Department, 

St.  Louis,  Mo. 

With  an  Introduction 

By 

H.  N.  Spencer,  M.  D.,  LL.  D. 

Professor  of  Otology,  Medical  Department  Washington  University, 

St.  Louis,  Mo. 

67  PAGES  OF  TEXT 
5  FULL  PAGE  PLATES 
12  OTHER  ILLUSTRATIONS 
PRICE  $1.00 

CONTENTS. 

Methods  of  Procedure  (General  Consideration). 

The  External  Ear. 

Diseases  of  the  Canal. 

The  Middle  Ear. 

The  Middle  Ear  (Continued),  Non-Suppurative  Conditions. 

The  Middle  Ear  (Continued),  Post-Suppurative  Conditions. 

The  Middle  Ear  (Continued),  Suppurative  Conditions. 

The  Middle  Ear  (Continued),  Purulent  Otitis  Media. 

The  Middle  Ear  (Continued),  Purulent  Otitis  Media. 

The  Middle  Ear  (Continued),  Operations    in    Chronic    Purulent    Otitis 

Media. 
The  Internal  Ear. 
Hearing  Tests. 
Intra-Cranial  Complications. 
Exercises  in  the  Surgical  Anatomy  of  the  Temporal  Bone. 

THE  C.   V.    MOSBY  MEDICAL 
BOOK  AND  PUBLISHING  CO. 

Grand  Avenue  and  Olive  Street,  St.  Louis,  Missouri. 


Chapter 

I. 

Chapter 

II. 

Chapter 

III. 

Chapter 

IV. 

Chapter 

V. 

Chapter 

VI. 

Chapter 

VII. 

Chapter 

VIII. 

Chapter 

XI. 

Chapter 

X. 

Chapter 

XI. 

Chapter 

XII. 

Chapter 

XIII. 

Chapter 

XIV. 

Suggestive  Therapeutics, 
Applied  Hypnotism 

and  Psychic  Science 

By  H.  S.  MUNRO,  A.  M.,  M.  D. 

Americus,  Ga. 
376  Pages,  Octavo.       Price,  S3. 00.       Sent  anywhere  upon  receipt  of  price. 


Publisher's  Announcement. 

That  suggestion  is  an  important  factor  in  the  treatment  of  diseases  is  no  longer 
denied  bj'  those  that  keep  abreast  of  medical  progress.  The  medical  journals  are  replete 
with  articles  from  the  leading  alienists  and  internists  of  this  country.  The  profession 
in  Europe  has  been  alive  to  the  importance  of  this  subject  for  many  years  and  much 
has  been  written  on  it  by  svich  men  as  Schofield,  Bernheim,  Forell  and  Duboise.  These 
writings  have  been  in  the  nature  of  research  work  and  have  not  been  devoted  to  the 
practical  application  of  this  branch  of  therapeutics  in  everyday  practice.  The  book 
herein  described  is  designed  to  give  a  practical  working  guide  to  the  general  practitioner 
in  the  application  of  suggestive  therapeutics.  Its  purpose  is  to  aid  the  profession  in 
reaching  a  correct  understanding  of  a  subject  that  has  been  shrouded  in  mj'stery  and 
used  by  the  cjuack  and  charlatan  in  many  cases  to  discredit  scientific  medicine.  The 
indorsements  that  have  been  given  the  author  by  prominent  phj-sicians  and  surgeons 
are  the  best  recommendations  the  book  can  have. 

CONTENTS. 

Introduction. 

Suggestion:  Its  Uses  and  Abuses. 

Hypnotism:  A     Demonstration     of    the     EfBciencj^    of     Suggestion. 

Technicjue  of  Inducing  the  Hj-pnotic  State. 
Theorj'  and  Practice  of  Suggestive  Therapeutics. 
Simple  Suggestions,  or  Suggestion  ^\'ithout  Hj-pnotism. 
Hj-pnotic    Suggestive    Therapeutics    Applied    in    3Iedicine,    Surgery. 
The  Psychological  Factor  in  Ob.stetrics. 
Training  the  Subconscious  Self  for  Health  and  Strength. 
Correct  Diagonsis  a  Safeguard  Against  Blunders. 
Philosoph}^  and  Religion  and  Their  Relation  to  Health. 
Conservation  of  Energj',  Education  and  Control  of  Emotions. 

Breathing,  Relaxation,  Dietetics,  Exercise,  etc. 
Roughing  It  as  a  Means  of  Health. 
Are  All  Specialists  Egotists? 
Personality  as  a  Factor  in  Therapeutics. 
Environment:  Its  Influence  in  Therapeutics. 
Brutality  of  Frankne-ss:   Hone.stj'  Imperative. 

Physical  and  Mental  Hygiene;   Character  as  a  Resource  of  Health. 
Suggestion  in  Education,  Character  Building,  etc. 
Moral  Stamina  a  Therapeutic  Power;  The  Higher  Art  in  Therapeutics, 

and  the  True  Physician. 
Chapter        XX.     Self-Mastery  as  a  Fine  Art. 


Chapter 

I. 

Chapter 

II. 

Chapter 

III. 

Chapter 

IV. 

Chapter 

V. 

Cahpter 

VI. 

Chapter 

VII. 

Chapter 

VIII. 

Chapter 

IX. 

Chapter 

X. 

Chapter 

XI. 

Chapter 

XII. 

Chapter 

XIII. 

Chapter 

XIV. 

Chapter 

XV. 

Chapter 

XVI. 

Chapter 

XVII. 

Chapter 

XVIII. 

Chapter 

XIX. 

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Grand  Ave.  and  Olive  Street,  St.  Louis,  Mo. 


Arteriosclerosis 


ETIOLOGY,     DIAGNOSIS,     PROGNOSIS,    PROPHYLAXIS    AND 

TREATMENT 

By 

L.  M.  Warfield,  A.  M.,  M.  D. 

With  an  Intrcducticn  by 

H.  S.  Thayer,  Baltimore,  Md. 

8  ORIGINAL  ILLUSTRATIONS. 


150  PAGES. 


PRICE  $2.00 


PUBLISHER'S  ANNOUNCEMENT 


This  book  is  particularly  opportune.  Tlie  rapid  pace  which  American.s  are  living, 
the  worrj^  and  mental  strain  under  which  the  majority  of  their  time  is  spent,  has  made 
this  a  nation  of  arterio-sclerotics.  The  author  has  laid  stress  upon  prophylaxis  as  well 
as  given  the  most  rational  treatment  known  to  modern  times.  The  text  is  embellished 
with  instructive  original  illustrations.      Sent  anywhere  on  receipt  of  price. 


Diseases  of  The  Skin 

By 

A.  H.  Ohmann-Dumesnil,  A.  M.,  M.  E.,  M.  D.,  Ph.  D.,  etc. 

Formerly  Professor  of  Dermatology  and  Syphilology  in  the  St.  Louis  College  for  .Medical 
Practioners;   the   St.   Louis   College  of   Physicians  and   Surgeons;  the   Marion- 
Sims  College  of  Medicine;  Member  of  the  St.  Louis  Medical  Society, 
of  the  Missouri  State  Medical  Association,  of  the  American 
Medical   Association,   of  the   1st,   2d,   3d,   4th,   5th 
and   6th   International    Dermatological 
Congress,  etc. 

THIRD  EDITION 

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PREFACE 

This  book  is  not  a  treatise.  The  intention  has  been  to  make  of  it  a  practical  guide 
to  the  easy  recognition  of  skin  diseases,  as  well  as  to  their  successful  treatment.  The 
remedies  which  have  been  recommended  are  such  as  may  be  found  in  every  practitian  s 
armamentarium  nwdicinorum.  ISo  attempt  has  been  made  to  write  an  elaborate  work, 
but  rather  to  furnish,  in  a  clear,  concise  manner,  just  that  information  most  desired  by 
medical  students  and  general  practitioners. 


TABLE  OF  CONTENTS. 


Prognosis. 

Symptomatology. 

Classifications. 

Diet  in   Skin   Di.seascs. 

Food  Eruptions. 

Aj^pendix. 


C.  V.  MOSBY  MEDICAL  BOOK  AND  PUBLISHING  CO. 

Grand  Avenue  and  OUve  Street,  ::  ::  St.  Louis,  Missouri. 


Chapter 

L 

The  Skin. 

Chapter 

VIII. 

Chapter 

IL 

Anatomy. 

Chapter 

IX. 

Chapter 

in. 

Physiology. 

Chapter 

X. 

Chapter 

IV. 

Diagnosis. 

Chapter 

XL 

Chapter 

V. 

Etiolog3^ 

Chapter 

XII. 

Chapter 

VI. 

Pathology. 

Chapter 

XIII. 

Chapter 

VII. 

Therapeutics. 

Office  Treatment  of  Rectal  Diseases 


By 


R.  D.  Mason,  M.  D. 


Professor  of  Rectal  Diseases  in  the  Creighton   University, 
Omaha,  Nebraska. 


New  4th  Edition.  367  Pages.  87  Illustrations.  Price  $2.50 

Tuberculosis  of  the  Nose  and  Throat 

By 

Lorenzo  B.  Lockord,  A.  B.,  M.  D. 
Denver,  Colorado 

Consulting  Laryngologist  to  the  Agnes  Memorial   Hospital,  Denver. 

504  Pages.  85  Illustrations.  64  of  which  are  colored.  Price  $5.00 


THE  C.  V.  MOSBY  MEDICAL  BOOK  AND  PUBLISHING  CO. 

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Hand  Book  of  Rectal  Diseases 

By 

L.  J.  Hirschman,  M.  D. 

Professor  of  Clinical  Proctology  Detroit  College  of  Medicine  and  Surger3^ 

150  Illustrations 
400  Pages.  Including  2  colored  Plates.  Price  $4.00 


Gonorrhea  in  Women 

By 
Palmer  Findley,  M.  D. 

Professor  of  Gynecology  in  the  Medical  Department  of  the  University  of  Nebraska, 

Omaha,   Nebraska. 

128  Pages.     Royal  Octavo.     Price  $2.00 


Chronic  Constipation 

By 
J.  A.  McMillian,  M.  D. 

Professor  of  Therapeutics  in  the  Detroit  College  of    Medicine  and  Surgery, 

Detroit,   Michigan. 

257  Pages.     Price  $2.00. 


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UNIVERSITY  OF  CALIFORNIA  LIBRARY 

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This  book  is  DUE  on  the  last  date  stamped  below. 


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